ANTXR2 Antibody

What is ANTXR2 and why is it significant in research?

ANTXR2 (anthrax toxin receptor 2), also known as CMG2 (capillary morphogenesis gene 2 protein), is a transmembrane protein that functions as a cellular receptor for anthrax toxin and plays important roles in extracellular matrix interactions. The protein contains an extracellular von Willebrand factor A (vWA) domain with a metal ion-dependent adhesion site (MIDAS) motif, an Ig-like domain, a single transmembrane helix, and a cytoplasmic tail . ANTXR2 has a molecular weight of approximately 54 kDa, though the observed weight in experiments is typically around 55 kDa .

ANTXR2 is significant in research for several key reasons. First, it serves as the primary receptor for protective antigen (PA), a key component of anthrax toxin from Bacillus anthracis, with binding affinity that is at least a thousand times greater than that of ANTXR1 . Once bound, PA facilitates the delivery of the other two anthrax toxin proteins, edema factor (EF) and lethal factor (LF), into the cytosol . Second, ANTXR2 plays crucial roles in capillary morphogenesis and maintaining the structure of basement membranes . Mutations in the ANTXR2 gene are associated with infantile systemic hyalinosis (ISH) and juvenile hyaline fibromatosis (JHF), rare stiff-skin syndromes characterized by hyaline deposition in various organs .

Research on ANTXR2 spans multiple disciplines, including infectious disease, developmental biology, and cancer research. Understanding its structure, expression patterns, and function provides important insights into both pathogen interactions and normal physiological processes.

What types of ANTXR2 antibodies are available for research and what are their specifications?

There are several types of ANTXR2 antibodies available for research applications, including polyclonal and monoclonal antibodies with various specificities and applications. These generally fall into the following categories:

Polyclonal Antibodies:

• Generated in hosts such as rabbits (e.g., 16723-1-AP from Proteintech)

• Target multiple epitopes of the ANTXR2 protein

• Often used for applications requiring high sensitivity

• Example specifications include rabbit IgG targeting human ANTXR2 with applications in Western blot (1:500-1:1000 dilution), immunoprecipitation (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate), and immunohistochemistry (1:50-1:500 dilution)

Monoclonal Antibodies:

• Generated from single B-cell clones (e.g., EPR7717 from Abcam)

• Target specific epitopes with high specificity

• Useful for applications requiring consistent results across experiments

• Example includes rabbit recombinant monoclonal antibodies suitable for IHC-P, ICC/IF, and WB applications

The following table summarizes key specifications for selected ANTXR2 antibodies:

When selecting an ANTXR2 antibody, researchers should consider the specific application, target species, and experimental conditions to ensure optimal results .

In which tissues and cell types is ANTXR2 expressed?

ANTXR2 exhibits a specific expression pattern across various tissues and cell types, which is important to consider when designing experiments targeting this protein. Based on immunohistochemistry and transcriptional analysis data, ANTXR2 expression has been characterized in the following tissues and cell types:

Human Tissues:

• Ovary tissue (positive IHC detection)

• Kidney tissue (positive IHC detection)

• Placenta tissue (positive IHC detection)

• Testis tissue (positive IHC detection)

• Lung tissue (positive IHC detection)

• Dorsal root ganglia (DRG) neurons (higher expression compared to CNS tissues)

Cell Types:

• T cells

• B cells

• Dendritic cells

• Monocytes

• Granulocytes

• Small- and medium-diameter neurons in the DRG

• HEK-293 cells (positive WB and IP detection)

• PC-3 cells (positive WB detection)

In neuronal tissues, ANTXR2 shows a distinctive expression pattern with high expression in the peripheral nervous system but limited expression in the central nervous system. RNA sequencing and in situ hybridization studies have shown that ANTXR2 is predominantly expressed in DRG neurons, particularly in nociceptors, while being largely absent in the spinal cord and brain regions . Within the human DRG, ANTXR2 is widely expressed in somatosensory neurons, including Calca+, P2rx3+, and Calca+/P2rx3+ neuronal populations .

For vascular research, it's notable that ANTXR2 was originally identified as a gene upregulated during capillary morphogenesis of endothelial cells cultured in vitro, and immunohistochemical studies have confirmed its expression in murine and human vasculature in vivo . This expression pattern supports its role in angiogenic regulation, particularly in endothelial proliferation and capillary morphogenesis.

What are the optimal protocols for using ANTXR2 antibodies in Western blot applications?

When using ANTXR2 antibodies for Western blot (WB) applications, researchers should follow a methodological approach to ensure reliable and reproducible results. Based on validated protocols and technical specifications for ANTXR2 antibodies, the following optimized WB protocol is recommended:

Sample Preparation:

• Prepare cell or tissue lysates in an appropriate lysis buffer containing protease inhibitors.

• Determine protein concentration using a standard method (e.g., BCA assay).

• Prepare 20-40 μg of total protein per lane in sample buffer containing a reducing agent.

• Heat samples at 95°C for 5 minutes to denature proteins.

Gel Electrophoresis and Transfer:

• Load samples on 8-12% SDS-PAGE gels (ANTXR2 has an expected molecular weight of approximately 54-55 kDa) .

• Run gel at 100-120V until desired separation is achieved.

• Transfer proteins to PVDF or nitrocellulose membrane (PVDF is often preferred for higher protein binding capacity).

• Confirm transfer efficiency with Ponceau S staining.

Antibody Incubation and Detection:

• Block membrane with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Incubate with primary ANTXR2 antibody at the recommended dilution (typically 1:500-1:1000 for polyclonal antibodies) .

• Incubate overnight at 4°C with gentle agitation.

• Wash 3-4 times with TBST, 5-10 minutes per wash.

• Incubate with appropriate HRP-conjugated secondary antibody (e.g., goat anti-rabbit IgG for rabbit primary antibodies) at 1:2000-1:5000 dilution for 1 hour at room temperature.

• Wash 3-4 times with TBST, 5-10 minutes per wash.

• Develop using an enhanced chemiluminescence (ECL) system.

Troubleshooting Tips:

• If background is high, increase blocking time or use 5% BSA instead of milk.

• If signal is weak, increase antibody concentration or protein loading.

• ANTXR2 may show glycosylation patterns resulting in multiple bands; Endoglycosidase H (EndoH) treatment can be used to assess glycosylation status .

• Validated positive controls include HEK-293 cells and PC-3 cells for human ANTXR2 .

When interpreting results, researchers should note that ANTXR2 typically appears at approximately 55 kDa , though variations may occur due to post-translational modifications or different isoforms. Experimental validation using ANTXR2 knockout or knockdown samples is recommended for confirming antibody specificity.

How can I optimize immunohistochemistry protocols for ANTXR2 detection in different tissue types?

Optimizing immunohistochemistry (IHC) protocols for ANTXR2 detection requires careful consideration of tissue type, fixation method, antigen retrieval, and antibody concentration. The following comprehensive protocol incorporates best practices for detecting ANTXR2 in various tissue types:

Tissue Preparation and Fixation:

• Fix tissues in 10% neutral buffered formalin for 24-48 hours.

• Process and embed tissues in paraffin.

• Section tissues at 4-5 μm thickness and mount on positively charged slides.

• Dry sections overnight at 37°C.

Antigen Retrieval (Critical for ANTXR2 Detection):

• Deparaffinize sections in xylene and rehydrate through graded alcohols to water.

• Primary recommended method: Heat-induced epitope retrieval using TE buffer pH 9.0 .

  • Immerse slides in TE buffer (10 mM Tris, 1 mM EDTA, pH 9.0).

  • Heat in a pressure cooker or microwave until boiling, then maintain at sub-boiling temperature for 15-20 minutes.

• Alternative method: Citrate buffer pH 6.0 .

  • This alternative may be necessary for certain tissue types or when encountering detection issues.

Immunostaining Protocol:

• Cool slides to room temperature and wash in PBS.

• Block endogenous peroxidase activity with 3% H₂O₂ in methanol for 10 minutes.

• Wash in PBS, 3 times for 5 minutes each.

• Block non-specific binding with 5% normal serum (from the same species as the secondary antibody) in PBS for 30 minutes.

• Incubate with primary ANTXR2 antibody at the optimal dilution:

  • For polyclonal antibodies like 16723-1-AP, use 1:50-1:500 dilution range .

  • For CAB6526, use 1:50-1:200 dilution range .

  • Incubate overnight at 4°C in a humidified chamber.

• Wash in PBS, 3 times for 5 minutes each.

• Apply appropriate biotinylated secondary antibody for 30 minutes at room temperature.

• Wash in PBS, 3 times for 5 minutes each.

• Apply avidin-biotin complex (ABC) for 30 minutes.

• Develop with DAB substrate.

• Counterstain with hematoxylin, dehydrate, clear, and mount.

Tissue-Specific Considerations:

• Human ovary, kidney, placenta, testis, and lung tissues have been validated as positive controls for ANTXR2 IHC .

• Mouse eye and rat lung tissues have also been confirmed as positive samples for certain ANTXR2 antibodies .

• For neuronal tissues (particularly DRG), extend the primary antibody incubation time to 48 hours at 4°C to improve penetration .

• For vascular studies, optimize the protocol to detect endothelial expression by using thinner sections (3 μm) and longer antigen retrieval times .

Validation and Controls:

• Include a negative control by omitting the primary antibody or using isotype control.

• Run a positive control tissue known to express ANTXR2.

• Consider dual immunofluorescence staining with cell-type-specific markers to confirm cellular localization.

• For validating neuronal expression, co-staining with neuronal markers like NeuN or PGP9.5 is recommended .

By following this optimized protocol and incorporating tissue-specific considerations, researchers can achieve reliable and specific detection of ANTXR2 in various tissue types for both research and diagnostic applications.

What are the most reliable methods for validating ANTXR2 antibody specificity?

Validating antibody specificity is critical for ensuring reliable research results. For ANTXR2 antibodies, several complementary approaches should be employed to comprehensively validate specificity:

Genetic Approaches for Validation:

• CRISPR/Cas9 Knockout (KO) Controls: Generate ANTXR2 knockout cell lines using CRISPR/Cas9 technology. These provide the most stringent negative controls for antibody validation. Several studies have utilized ANTXR2 KO models, demonstrating complete loss of signal with specific antibodies .

• RNA Interference: Use siRNA or shRNA to knockdown ANTXR2 expression. While not as complete as CRISPR knockout, significant reduction in signal (>70%) should be observed in knockdown samples compared to controls . An example RT-PCR protocol for verifying knockdown:

  • Extract total RNA using RNeasy kit

  • Perform first strand synthesis with random hexamers and reverse transcriptase

  • Conduct qPCR with ANTXR2-specific primers:

    • Forward: 5′-CTCTTGCAAAAAAGCCTTCG-3′

    • Reverse: 5′-TTCTTTGCCTCGTTCTCTGC-3′

  • Normalize to a housekeeping gene like β-actin

• Overexpression Systems: Transfect cells with ANTXR2-expressing constructs and confirm increased signal intensity compared to control transfections.

Biochemical Validation Approaches:

• Western Blot Band Specificity: Verify that the antibody detects a protein of the expected molecular weight (54-55 kDa for ANTXR2) . Treatment with Endoglycosidase H can help confirm glycosylation patterns specific to ANTXR2 .

• Immunoprecipitation-Mass Spectrometry: Immunoprecipitate using the ANTXR2 antibody, then identify the pulled-down proteins using mass spectrometry to confirm that ANTXR2 is among the enriched proteins.

• Epitope Blocking: Pre-incubate the antibody with the immunizing peptide or recombinant ANTXR2 protein before applying to samples. This should abolish or significantly reduce specific staining.

Cross-Validation with Multiple Antibodies:

• Multiple Antibody Approach: Use at least two different antibodies targeting distinct epitopes of ANTXR2. Consistent staining patterns provide strong evidence for specificity.

• Cross-Species Reactivity Testing: If the antibody claims cross-reactivity with multiple species (e.g., human, mouse, rat), test samples from each species and compare the staining patterns and molecular weights.

Application-Specific Validation:

• For Immunohistochemistry/Immunofluorescence:

  • Compare staining patterns with known expression data

  • Use tissues with validated ANTXR2 expression (e.g., human ovary, kidney, placenta, testis, lung)

  • Perform dual labeling with established cell-type markers (e.g., CD31 for endothelial cells)

• For Flow Cytometry:

  • Include fluorescence-minus-one (FMO) controls

  • Compare expression patterns on known positive cell types (e.g., T cells, B cells, dendritic cells, monocytes, granulocytes)

• For Functional Assays:

  • Confirm that antibody-mediated effects are consistent with genetic manipulation of ANTXR2

  • Test for expected biological effects, such as inhibition of anthrax toxin binding

A systematic validation approach using multiple methods provides the strongest evidence for antibody specificity. Documentation of validation results is essential for rigorous research practices and should be included in methods sections of publications.

How can ANTXR2 antibodies be used to investigate the molecular mechanisms of anthrax toxin entry?

ANTXR2 antibodies provide powerful tools for investigating the complex molecular mechanisms of anthrax toxin entry into cells. Advanced methodological approaches using these antibodies can illuminate various aspects of this process:

Receptor-Toxin Interaction Studies:

• Co-immunoprecipitation (Co-IP) Assays: ANTXR2 antibodies can be used for Co-IP to study the interaction between ANTXR2 and protective antigen (PA), as well as associated proteins . The procedure involves:

  • Treating cells with PA or PA mutants

  • Lysing cells in appropriate buffers

  • Immunoprecipitating with ANTXR2 antibodies (recommended usage: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate)

  • Analyzing precipitates by Western blot for PA and potential cofactors

  • This approach has revealed critical insights into the role of the metal ion-coordinated by residue D683 of PA in receptor binding

• Surface Plasmon Resonance (SPR): Immobilize purified ANTXR2 ectodomain on a sensor chip and study real-time binding kinetics with various PA variants. ANTXR2 antibodies can be used to confirm the proper orientation and functionality of the immobilized receptor.

• Receptor Competition Assays: Use labeled ANTXR2 antibodies to quantify displacement by PA variants, providing insights into binding site specificity and affinity differences between ANTXR1 and ANTXR2 .

Receptor Trafficking and Internalization Studies:

• Live Cell Imaging: Combine ANTXR2 antibodies with fluorescently labeled PA to track:

  • Initial binding at the cell surface

  • Movement to lipid rafts

  • Clathrin-dependent internalization

  • Endosomal trafficking

• Endocytosis Assays: Antibodies against ANTXR2 can help quantify receptor internalization rates:

  • Label cell surface ANTXR2 with antibodies at 4°C

  • Allow internalization at 37°C for various time periods

  • Remove remaining surface antibodies with acid wash

  • Quantify internalized antibody by microscopy or flow cytometry

• pH-Dependent Conformational Changes: Using conformation-specific ANTXR2 antibodies to detect structural changes that occur during endosomal acidification, which are critical for PA pore formation.

Genetic and Structural Studies:

• Epitope Mapping: Use a panel of ANTXR2 antibodies targeting different domains to determine critical regions for toxin binding and internalization. This can be done through:

  • Competitive binding assays

  • Mutagenesis of suspected epitopes

  • ANTXR2 domain truncation experiments

• SNP Analysis: ANTXR2 antibodies can help correlate genetic polymorphisms with functional outcomes:

  • Generate cell lines expressing ANTXR2 variants (e.g., those with SNPs rs13140055 and rs80314910 that affect promoter activity)

  • Use antibodies to quantify receptor expression levels

  • Correlate expression with toxin susceptibility

  • This approach has helped establish that genetic variations in ANTXR2 significantly impact anthrax toxin sensitivity

Advanced Therapeutic Applications:

• Neutralizing Antibody Development: Screen for ANTXR2 antibodies that can:

  • Block PA binding to prevent toxin entry

  • Inhibit receptor conformational changes required for pore formation

  • Prevent receptor trafficking to endosomes

• Receptor Decoy Strategies: Use ANTXR2 antibodies to validate the efficacy of soluble receptor decoy inhibitors (RDI) consisting of the extracellular domain of ANTXR2, which can neutralize even antibody-resistant forms of anthrax toxin .

• Targeted Delivery Systems: Leveraging the ANTXR2-PA interaction to develop targeted therapeutic delivery systems, with antibodies used to confirm specific targeting .

By employing these advanced methodological approaches with ANTXR2 antibodies, researchers can gain deeper insights into the intricate molecular mechanisms of anthrax toxin entry and develop potential therapeutic interventions.

What role does ANTXR2 play in angiogenesis and how can antibodies help elucidate this function?

ANTXR2 (originally identified as Capillary Morphogenesis Gene 2 or CMG2) plays significant roles in angiogenesis beyond its function as an anthrax toxin receptor. Antibody-based approaches provide powerful tools for investigating these angiogenic functions through multiple experimental paradigms:

Characterization of ANTXR2 Expression in Vascular Contexts:

• Immunohistochemical Profiling: ANTXR2 antibodies enable the mapping of expression patterns in vascular structures across normal and pathological tissues. Studies have confirmed ANTXR2 expression in both murine and human vasculature in vivo, with expression not restricted solely to endothelial cells . This technique requires:

  • Optimal antigen retrieval (TE buffer pH 9.0 recommended)

  • Appropriate antibody dilution (1:50-1:200 for IHC)

  • Co-staining with endothelial markers (CD31, von Willebrand factor)

  • Quantitative image analysis of vascular density and ANTXR2 co-localization

• Flow Cytometric Analysis: Using fluorescently-labeled ANTXR2 antibodies to quantify expression levels on:

  • Different endothelial cell populations (arterial vs. venous; macro vs. microvascular)

  • Endothelial progenitor cells

  • Pericytes and supporting vascular cells

  • Comparison between quiescent and activated endothelial states

Functional Analyses of ANTXR2 in Angiogenic Processes:

• In Vitro Angiogenesis Assays: ANTXR2 antibodies can be employed in multiple ways to assess functional roles:

  • As tools to confirm knockdown efficiency in RNA interference experiments

  • As potential inhibitors of ANTXR2 function when function-blocking antibodies are used

  • As detection reagents in proteomic analyses of ANTXR2-associated complexes

  Research has demonstrated that reduced ANTXR2 expression via RNA interference significantly inhibits endothelial cell proliferation and impairs their capacity to form capillary-like networks in vitro . Specific experimental approaches include:

  • MTT or BrdU proliferation assays following ANTXR2 manipulation

  • Matrigel tube formation assays to assess morphogenesis

  • Scratch wound migration assays to evaluate motility

  • Spheroid sprouting assays to model 3D angiogenic responses

• Extracellular Matrix Interaction Studies: ANTXR2 interacts with basement membrane components, particularly laminin and collagen type IV . Antibodies facilitate investigation of these interactions through:

  • Co-immunoprecipitation of ANTXR2 with matrix proteins

  • Proximity ligation assays to detect in situ protein associations

  • Immunofluorescence co-localization analyses

  • ANTXR2 blocking experiments to assess adhesion to specific matrix components

• Molecular Signaling Analysis: Examine how ANTXR2 engagement affects angiogenic signaling pathways:

  • Phosphorylation states of key angiogenic mediators after ANTXR2 manipulation

  • Transcriptional responses using reporter assays

  • Proteomic analysis of ANTXR2 immunoprecipitates to identify novel interacting partners

Advanced In Vivo Angiogenesis Models:

• Tumor Angiogenesis: ANTXR2 antibodies enable evaluation of expression in tumor vasculature:

  • Quantification of vessel density and ANTXR2 co-expression

  • Correlation with markers of vascular maturation/permeability

  • Potential therapeutic targeting of tumor vessels expressing ANTXR2

• Developmental Angiogenesis: Study ANTXR2's role in embryonic and postnatal vessel formation:

  • Whole-mount immunostaining of developing vascular networks

  • Temporal expression patterns during key angiogenic periods

  • Correlation with vessel sprouting, stabilization, and pruning events

• Tissue-Specific Angiogenic Responses: Using conditional knockout models (e.g., Cdh5-cre/Antxr2 fl/fl for endothelial-specific deletion) combined with antibody detection to study:

  • Wound healing angiogenesis

  • Hypoxia-induced neovascularization

  • Inflammation-associated angiogenesis

Translational Implications:

• Biomarker Development: Validation of ANTXR2 as a potential biomarker for:

  • Tumor angiogenesis progression

  • Vascular malformations

  • Therapeutic response to anti-angiogenic interventions

• Therapeutic Targeting: Exploration of ANTXR2-targeting strategies:

  • Development of function-blocking antibodies

  • Antibody-drug conjugates for vascular-specific delivery

  • Monitoring therapy response through changes in ANTXR2 expression

The methodological approaches outlined above provide a comprehensive framework for investigating ANTXR2's angiogenic functions, potentially revealing new therapeutic targets for vascular disorders and cancer.

How can researchers investigate ANTXR2 genetic polymorphisms and their impact on protein expression and function?

Investigating the relationship between ANTXR2 genetic polymorphisms and their functional outcomes requires a multidisciplinary approach combining genomic, transcriptomic, proteomic, and functional analyses. Advanced methodological strategies using ANTXR2 antibodies can help elucidate these complex genotype-phenotype relationships:

Genomic Analysis and Polymorphism Identification:

• SNP Screening and Selection: Begin by identifying relevant ANTXR2 SNPs through:

  • Database mining from resources like dbSNP, gnomAD, or 1000 Genomes Project

  • Focus on functional SNPs in regulatory regions (e.g., rs13140055 and rs80314910 in the promoter) and coding regions (e.g., rs12647691)

  • Prioritize SNPs with potential functional impact using in silico prediction tools like PolyPhen-2, SNAP2, and others as described in recent studies

• Genotyping Methodologies:

  • Targeted sequencing of ANTXR2 gene regions

  • Custom SNP arrays or multiplex PCR approaches

  • Whole genome/exome sequencing for comprehensive variant detection

Transcriptional Impact Assessment:

• Allele-Specific Expression Analysis:

  • Use quantitative RT-PCR to measure ANTXR2 transcript levels in samples with different SNP genotypes

  • Employ allele-specific primers to distinguish expression from different alleles

  • RNA-seq to assess global expression changes associated with specific genotypes

• Promoter Activity Assays:

  • Clone ANTXR2 promoter variants containing different SNP combinations into luciferase reporter vectors

  • Transfect into relevant cell types and measure promoter activity

  • Studies have demonstrated that promoter variants with G at rs13140055 and CTT at rs80314910 result in elevated ANTXR2 expression

• Transcription Factor Binding Analysis:

  • Perform electrophoretic mobility shift assays (EMSA) to assess how SNPs affect transcription factor binding

  • Chromatin immunoprecipitation (ChIP) to examine binding of relevant factors like CREB to variant sequences

  • DNA affinity precipitation assays to identify differential protein binding to SNP-containing sequences

Protein Expression and Localization Analysis:

• Quantitative Protein Analysis Using ANTXR2 Antibodies:

  • Western blot analysis of ANTXR2 protein levels in cells/tissues with different genotypes

  • Flow cytometry to quantify cell surface expression of ANTXR2 across genotypes

  • Immunohistochemistry to assess tissue-specific expression patterns

  • These approaches can validate whether genomic variations translate to altered protein levels

• Surface Biotinylation Experiments:

  • Label cell surface proteins with biotin

  • Immunoprecipitate with streptavidin beads

  • Detect ANTXR2 by Western blot using specific antibodies

  • Quantify surface expression relative to total cellular ANTXR2

  • This technique has been successfully employed to measure ANTXR2 trafficking differences

• Subcellular Localization Studies:

  • Immunofluorescence microscopy to examine potential differences in ANTXR2 localization between variant forms

  • Co-localization with organelle markers to assess trafficking patterns

  • Live-cell imaging to monitor dynamic changes in receptor distribution

Functional Impact Assessment:

• Toxin Sensitivity Assays:

  • Expose cells expressing different ANTXR2 variants to anthrax lethal toxin

  • Measure cell viability, morphological changes, or specific toxin-induced cellular responses

  • Correlate toxin sensitivity with genotype and expression levels

  • Studies have demonstrated statistically significant correlations between specific SNP combinations and anthrax toxin sensitivity

• Receptor-Ligand Binding Studies:

  • Surface plasmon resonance (SPR) to measure binding affinity between PA and different ANTXR2 variants

  • Co-immunoprecipitation to assess complex formation efficiency

  • FRET-based interaction assays to examine binding dynamics

  • The non-synonymous SNP rs12647691 has been shown to affect binding affinity between anthrax toxin and its receptor

• Structural Impact Analysis:

  • Computational modeling of how coding variants affect protein structure

  • Thermal stability assays to assess structural integrity differences

  • Limited proteolysis to probe conformational variations

Integrative Approaches:

• CRISPR-Based Genomic Editing:

  • Generate isogenic cell lines differing only in ANTXR2 SNP genotypes

  • Perform comprehensive phenotypic analyses on these lines

  • This approach controls for genetic background effects that might confound association studies

• 3C (Chromatin Conformation Capture) Technology:

  • Investigate how promoter SNPs affect long-range chromosomal interactions

  • Studies have identified a region of 3550 bp in length located 70 kb upstream to the transcription start site that spatially co-localizes with the promoter region

• Combined Genetic Risk Assessment:

  • Analyze the combinatorial effects of multiple SNPs on ANTXR2 function

  • For example, combining regulatory SNPs (rs13140055 and rs80314910) with the coding SNP (rs12647691) provides a more comprehensive view of genetic influences on anthrax toxin sensitivity

This multi-layered approach to investigating ANTXR2 polymorphisms enables researchers to build a comprehensive understanding of how genetic variations influence protein expression, localization, and function, with potential implications for individual differences in disease susceptibility and therapeutic responses.

What are the emerging applications of ANTXR2 antibodies in targeted therapeutic delivery systems?

The unique expression pattern and internalization properties of ANTXR2 make it an attractive target for developing advanced therapeutic delivery systems. ANTXR2 antibodies play crucial roles in the development and validation of these emerging applications:

ANTXR2-Targeted Drug Delivery Platforms:

• Antibody-Drug Conjugates (ADCs):

  • ANTXR2 antibodies can be directly conjugated to cytotoxic payloads

  • Particularly promising for targeting tissues with high ANTXR2 expression

  • Validation requires demonstrating specific binding, internalization, and selective cytotoxicity

  • Quantitative assessment of target expression using immunohistochemistry and flow cytometry is essential for patient selection

• Protective Antigen (PA)-Based Delivery Systems:

  • Leveraging the natural PA-ANTXR2 interaction for payload delivery

  • Modified PA proteins can act as delivery vehicles for therapeutic cargo

  • ANTXR2 antibodies are crucial for validating target expression and specificity

  • This approach has been successfully used to deliver molecular cargo into the cytoplasm of specific cell populations

• Nanoparticle Targeting:

  • ANTXR2 antibodies or antigen-binding fragments can be conjugated to nanoparticles

  • Enables targeted delivery of various therapeutic modalities (small molecules, nucleic acids, proteins)

  • Multi-modal imaging agents can be incorporated for theranostic applications

  • Surface modification with ANTXR2-targeting moieties enhances cellular uptake in ANTXR2-expressing tissues

Targeting ANTXR2 in Specific Therapeutic Contexts:

• Pain Management Applications:

  • ANTXR2 is highly expressed in somatosensory neurons, particularly nociceptors, with limited expression in CNS tissues

  • This distinctive expression pattern makes it an ideal target for pain-specific interventions

  • Modified anthrax toxin components have been used to regulate pain signaling through ANTXR2

  • Antibodies are essential for validating:

    • Target expression in relevant neuronal populations

    • Penetration of targeted agents into pain-transmitting neurons

    • Confirmation of mechanism through immunohistochemical analysis of neuronal subtypes

• Cancer-Directed Therapies:

  • ANTXR2 expression has been identified in tumor vasculature through immunohistochemical studies

  • Antibody-based targeting can be directed at:

    • Tumor angiogenesis through endothelial cell targeting

    • Direct targeting of ANTXR2-expressing tumor cells

    • Tumor microenvironment modulation

  • Methodologies include:

    • Patient-derived xenograft models to validate targeting efficiency

    • Immunohistochemical profiling of tumors for patient selection

    • Biodistribution studies using labeled antibodies to confirm tumor localization

• Modulating Fibrotic Disorders:

  • ANTXR2 mutations are associated with hyaline fibromatosis syndromes

  • Targeted delivery of anti-fibrotic agents to ANTXR2-expressing cells

  • Immunohistochemical mapping of ANTXR2 in fibrotic tissues guides therapy development

  • Antibodies can help validate disease mechanisms and therapeutic targeting

Advanced Delivery Strategies and Validation Methods:

• Engineered PA Variants with Enhanced Specificity:

  • Development of D683K mutant forms of PA that bind specifically to ANTXR2 but not ANTXR1

  • This specificity enables ANTXR2-exclusive targeting

  • Antibodies are critical for validating:

    • Selective binding to ANTXR2 versus ANTXR1

    • Internalization pathways

    • Functional payload delivery

• Receptor Decoy Strategies:

  • Soluble ANTXR2 ectodomains can neutralize toxins, suggesting potential therapeutic applications

  • Antibodies help characterize binding and neutralization efficiency

  • May serve as complementary approaches to antibody-based therapies

• Cell-Specific Targeting Validation:

  • Using genetic models (e.g., Advillin-creERT2/Antxr2 fl/fl for somatosensory neurons, Cdh5-cre/Antxr2 fl/fl for endothelial cells)

  • These models combined with antibody-based detection confirm:

    • Cell-type specific deletion

    • Therapeutic specificity

    • Mechanism of action

• Biodistribution and Safety Assessment:

  • Labeled ANTXR2 antibodies to track tissue distribution of targeted therapies

  • Immunohistochemical analysis of off-target binding

  • Assessment of internalization efficiency in target versus non-target tissues

Translational Considerations and Clinical Development:

• Patient Selection Biomarkers:

  • ANTXR2 antibodies for immunohistochemical screening of patient samples

  • Quantitative assessment of expression levels to predict response

  • Correlation of expression with genetic polymorphisms that might affect targeting efficacy

• Companion Diagnostic Development:

  • Standardized IHC protocols using validated ANTXR2 antibodies

  • Flow cytometry-based assays for circulating cells

  • Integration with genetic testing for relevant ANTXR2 polymorphisms

• Monitoring Therapeutic Response:

  • Serial sampling to assess changes in ANTXR2 expression during treatment

  • Development of imaging probes based on ANTXR2 antibodies for non-invasive monitoring

  • Correlation of expression changes with clinical outcomes

These emerging applications represent a frontier in targeted therapeutics, with ANTXR2 antibodies serving as essential tools for development, validation, and clinical implementation of these innovative approaches.

What are common technical challenges when working with ANTXR2 antibodies and how can they be resolved?

Researchers working with ANTXR2 antibodies may encounter several technical challenges that can affect experimental outcomes. Here are comprehensive solutions to common issues:

Non-specific Binding and High Background:

• Problem: High background staining in immunohistochemistry or Western blots.
  Solutions:

  • Optimize blocking conditions: Test different blocking agents (5% BSA often performs better than milk for ANTXR2 detection)

  • Increase blocking time to 2 hours at room temperature

  • Perform more stringent washing: Increase number of washes (5-6 times) and duration (10 minutes each)

  • Use 0.1-0.3% Triton X-100 in washing buffer to reduce non-specific hydrophobic interactions

  • Titrate primary antibody: Test dilution series from 1:50 to 1:1000 to determine optimal concentration

  • For polyclonal antibodies, consider pre-absorption with tissue lysates from ANTXR2-knockout samples

• Problem: Multiple bands in Western blot.
  Solutions:

  • Validate with positive controls (HEK-293 cells, PC-3 cells)

  • Use recombinant ANTXR2 protein as a standard

  • Note that ANTXR2 is glycosylated, which can result in multiple bands; Endoglycosidase H treatment can help confirm glycosylation-related bands

  • Use gradient gels (4-12%) for better resolution of closely spaced bands

  • Increase running time for better separation

Poor Signal Detection:

• Problem: Weak or absent signal in Western blot.
  Solutions:

  • Ensure proper sample preparation: Use RIPA buffer with protease inhibitors for efficient extraction

  • Avoid repeated freeze-thaw cycles of antibody and samples

  • Optimize protein loading: 40-60 μg total protein per lane may be necessary

  • Increase antibody concentration or incubation time (overnight at 4°C)

  • Use enhanced sensitivity detection systems (ECL Plus instead of standard ECL)

  • For membrane proteins like ANTXR2, avoid boiling samples (heat at 70°C for 10 minutes instead)

  • Verify transfer efficiency with reversible staining (Ponceau S)

• Problem: Weak signal in immunohistochemistry.
  Solutions:

  • Optimize antigen retrieval: TE buffer pH 9.0 is recommended for ANTXR2, with heat-induced epitope retrieval

  • Alternative antigen retrieval with citrate buffer pH 6.0 may work better for some tissue types

  • Extend primary antibody incubation to overnight at 4°C or even 48 hours for thick sections

  • Use signal amplification systems (tyramide signal amplification or polymer-based detection)

  • Ensure tissues are properly fixed (overfixation can mask epitopes)

  • Use freshly cut sections (epitope accessibility decreases in stored sections)

Inconsistent Results Between Experiments:

• Problem: Variable staining intensity between experiments.
  Solutions:

  • Standardize all protocol steps: temperature, incubation times, reagent concentrations

  • Prepare larger volumes of antibody dilutions to use across multiple experiments

  • Include positive control samples in every experiment

  • Process all comparative samples simultaneously

  • Document lot numbers of antibodies and other reagents

  • Consider monoclonal antibodies for more consistent results

• Problem: Differences between recombinant expression systems and endogenous expression.
  Solutions:

  • Use epitope tags distant from functional domains

  • Validate with multiple antibodies targeting different epitopes

  • Compare expression levels using quantitative methods

  • Consider native versus denatured protein conformation

Application-Specific Challenges:

• Problem: Poor immunoprecipitation efficiency.
  Solutions:

  • Optimize antibody amount (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate is recommended)

  • Use gentler lysis buffers to preserve protein-protein interactions

  • Pre-clear lysates to reduce non-specific binding

  • Cross-link antibody to beads to prevent antibody contamination in eluted samples

  • Consider using magnetic beads instead of agarose for cleaner isolation

• Problem: Inconsistent flow cytometry results.
  Solutions:

  • Optimize fixation and permeabilization protocols

  • Use Fc receptor blocking to reduce non-specific binding

  • Include viability dye to exclude dead cells

  • Establish consistent gating strategy

  • Include fluorescence-minus-one (FMO) controls

• Problem: Poor staining in neuronal tissues.
  Solutions:

  • Use specific fixation protocols optimized for neurons

  • Extend antibody incubation times (48 hours at 4°C)

  • Use tissue clearing techniques for better antibody penetration

  • Consider post-fixation with 4% paraformaldehyde after antigen retrieval

  • For DRG tissues, optimize sectioning thickness (10-12 μm recommended)

Validation and Quality Control Measures:

• Implement rigorous validation protocols:

  • Always include positive controls (confirmed ANTXR2-expressing cells/tissues)

  • Include negative controls (ANTXR2 knockout or knockdown samples)

  • Verify results with orthogonal methods (e.g., validate IHC with Western blot)

  • Document antibody validation data including lot number, dilution, and specific protocol conditions

• Antibody storage and handling:

  • Aliquot antibodies to avoid repeated freeze-thaw cycles

  • Store according to manufacturer recommendations (typically -20°C)

  • For polyclonal antibodies in glycerol, avoid diluting the stock solution

  • Check for precipitation before use and centrifuge if necessary

By systematically addressing these common technical challenges, researchers can significantly improve the reliability and reproducibility of experiments using ANTXR2 antibodies across different applications.

How do different tissue fixation and processing methods affect ANTXR2 antibody performance?

Tissue fixation and processing methods significantly impact ANTXR2 antibody performance in immunohistochemistry and immunofluorescence applications. Understanding these effects is crucial for obtaining reliable and reproducible results:

Impact of Fixative Type on ANTXR2 Epitope Preservation:

• Formalin Fixation:

  • 10% neutral buffered formalin (NBF) is most commonly used and generally compatible with ANTXR2 detection

  • Optimal fixation time: 24-48 hours for standard tissues; overfixation can mask epitopes

  • Mechanism: Forms methylene bridges between proteins that can conceal ANTXR2 epitopes

  • Requires heat-induced epitope retrieval (HIER) for optimal ANTXR2 detection

  • Recommended protocols specify TE buffer pH 9.0 or alternative citrate buffer pH 6.0 for antigen retrieval

• Paraformaldehyde (PFA) Fixation:

  • 4% PFA provides good epitope preservation with less extensive cross-linking

  • Preferred for immunofluorescence applications targeting ANTXR2

  • Shorter fixation times (4-24 hours) generally maintain better antigenicity

  • Still requires antigen retrieval but may be less stringent than formalin-fixed tissues

  • Particularly effective for neuronal tissues when studying ANTXR2 expression in DRG neurons

• Alcohol-Based Fixatives:

  • Methanol or ethanol fixation preserves different epitopes than aldehyde fixatives

  • May preserve certain conformational epitopes of ANTXR2 without requiring antigen retrieval

  • Produces poorer morphological preservation but can enhance membrane protein detection

  • Generally not recommended as primary fixative for ANTXR2 IHC but can be useful for immunocytochemistry

  • Can be used as post-fixation step after formalin fixation to enhance certain epitopes

• Frozen Tissue Preparation:

  • Preferable for preserving native protein conformation and certain epitopes

  • Rapid freezing in OCT compound followed by cryosectioning

  • Brief post-fixation (10 minutes) in 4% PFA or acetone recommended

  • May provide superior detection of membrane-localized ANTXR2

  • Challenges include poorer morphological preservation and potential freezing artifacts

Processing Parameters That Affect ANTXR2 Detection:

• Tissue Processing Time and Temperature:

  • Extended processing at high temperatures can denature ANTXR2 epitopes

  • Recommended: Use shorter processing protocols with lower temperatures

  • Microwave-assisted processing can reduce time while preserving antigens

  • Vacuum infiltration processors may improve reagent penetration and consistency

• Embedding Media Effects:

  • Paraffin embedding: Most common but requires complete paraffin removal

  • Low-temperature embedding compounds: Less epitope masking but poorer morphology

  • Specialized embedding media (e.g., hydrophilic resins): May preserve membrane proteins better

  • Paraffin quality and embedding temperature affect antigen preservation

• Section Thickness Considerations:

  • Optimal section thickness for ANTXR2 IHC: 4-5 μm for general tissues

  • For DRG and neuronal tissues: 10-12 μm sections may provide better signal

  • For detailed subcellular localization: 2-3 μm sections may be preferable

  • Thicker sections (>10 μm) may require extended antibody incubation times

Optimal Antigen Retrieval Methods for ANTXR2:

• Heat-Induced Epitope Retrieval (HIER):

  • Primary recommended buffer: TE buffer pH 9.0

  • Alternative buffer: Citrate buffer pH 6.0

  • Heating methods:

    • Pressure cooker: 3 minutes at full pressure (most effective)

    • Microwave: 20 minutes at sub-boiling temperature

    • Water bath: 30-40 minutes at 95-98°C

  • Critical factors: Buffer pH, temperature, time, and cooling method

  • Optimal protocol may vary between tissue types and antibody clones

• Enzymatic Retrieval:

  • Proteinase K or trypsin digestion can expose certain epitopes

  • Generally less effective than HIER for ANTXR2

  • May be useful as complementary approach for difficult tissues

  • Concentration and incubation time require careful optimization

• Combined Approaches:

  • Sequential application of HIER followed by mild enzymatic digestion

  • Dual buffer systems with different pH values applied sequentially

  • These approaches may recover different ANTXR2 epitopes for comprehensive detection

Tissue-Specific Optimization Strategies:

• Neuronal Tissues:

  • Require specialized handling due to high lipid content

  • Post-fixation with 4% PFA after antigen retrieval may help

  • Extended antibody incubation (48 hours at 4°C) for thick sections

  • Consider tissue clearing techniques for whole-mount preparations

• Highly Vascularized Tissues:

  • May require modified fixation to preserve endothelial integrity

  • Perfusion fixation recommended when possible

  • Shorter fixation times to prevent overfixation of vessel walls

  • Dual staining with endothelial markers helps confirm vascular ANTXR2 expression

• Fibrotic Tissues:

  • Dense collagen can impede antibody penetration

  • May require extended retrieval times

  • Pretreatment with hyaluronidase can improve antibody access

  • Particularly relevant when studying ANTXR2 in hyaline fibromatosis syndromes

Optimization Protocol for Challenging Samples:

• Fixation Comparison Test:

  • Process parallel samples with different fixatives (10% NBF, 4% PFA, methanol)

  • Compare ANTXR2 staining patterns and intensity

  • Select optimal fixative for specific application

• Antigen Retrieval Matrix Testing:

  • Create a matrix of retrieval conditions:

    • pH gradients (pH 6.0, 7.0, 9.0)

    • Time variations (10, 20, 30 minutes)

    • Different retrieval methods (pressure, microwave, enzyme)

  • Evaluate ANTXR2 signal intensity and specificity

  • Document optimal conditions for future reference

• Sequential Double Retrieval Approach:

  • Initial HIER with TE buffer pH 9.0 for 20 minutes

  • Cool to room temperature

  • Secondary HIER with citrate buffer pH 6.0 for 10 minutes

  • This approach may recover additional epitopes

By systematically evaluating these fixation and processing variables, researchers can develop optimized protocols for ANTXR2 detection in specific tissue types and experimental contexts, ensuring consistent and reliable antibody performance.

What are the latest findings regarding ANTXR2's role in pain signaling pathways?

Recent research has revealed an unexpected and significant role for ANTXR2 in pain signaling pathways, opening new avenues for pain research and therapeutic development. The following methodological approaches and findings highlight this emerging area:

Expression Profile of ANTXR2 in Pain-Sensing Neurons:

• Transcriptional Profiling of ANTXR2 in Somatosensory Neurons:

  • RNAseq analysis has demonstrated that ANTXR2 is highly expressed in dorsal root ganglia (DRG) neurons while being virtually absent from central nervous system (CNS) tissues, including the spinal cord and brain regions .

  • Single-cell transcriptomic studies have revealed that ANTXR2 is predominantly expressed in small- and medium-diameter neurons, which typically function as nociceptors .

  • Quantitative RT-PCR analysis has confirmed significantly higher ANTXR2 expression in DRG compared to various CNS regions, including the brain and spinal cord .

• Neuronal Subtype-Specific Expression Patterns:

  • RNAscope in situ hybridization analysis has shown that:

    • ANTXR2 is expressed in most SCN10a+ cells (encoding Nav1.8 sodium channels associated with nociceptors)

    • ANTXR2 is expressed in only a small subset of Pvalb+ proprioceptive neurons

    • In sorted DRG neuron populations, ANTXR2 is enriched in both IB4+ and IB4- Nav1.8 lineage neurons compared to parvalbumin lineage proprioceptors

  • These expression patterns suggest a specific role in pain-sensing neural circuits rather than mechanosensory pathways.

• Comparative Human and Mouse Expression Profiles:

  • RNAscope analysis of human DRG neurons has demonstrated that ANTXR2 is widely expressed in human somatosensory neurons, including:

    • Calca+ neurons (expressing calcitonin gene-related peptide)

    • P2rx3+ neurons (purinergic receptor-expressing neurons)

    • Calca+/P2rx3+ neurons (a major subset of nociceptors)

  • Size-based analysis confirmed that ANTXR2 is primarily expressed in small and medium diameter DRG neurons in humans

  • This conservation of expression pattern between species suggests an evolutionarily preserved role in pain signaling.

Functional Role of ANTXR2 in Pain Modulation:

Molecular Mechanisms of ANTXR2-Mediated Pain Signaling:

• Signaling Pathways Downstream of ANTXR2 Activation:

  • The anthrax edema toxin (ET) consists of protective antigen (PA) and edema factor (EF), with EF functioning as an adenylate cyclase that increases intracellular cAMP levels .

  • This suggests that ANTXR2-mediated analgesia may operate through cAMP-dependent signaling pathways in nociceptors.

  • Immunohistochemical co-localization studies with ANTXR2 antibodies and markers of specific signaling pathways can help elucidate the downstream mechanisms.

• Interaction with Established Pain Signaling Components:

  • Ongoing research is investigating how ANTXR2 activation modulates:

    • Voltage-gated ion channels critical for neuronal excitability

    • Transduction channels involved in detecting noxious stimuli

    • Neurotransmitter release from nociceptor terminals

  • Co-immunoprecipitation studies with ANTXR2 antibodies can help identify molecular interaction partners in pain signaling complexes.

Translational Implications for Pain Management:

• ANTXR2 as a Novel Target for Pain Therapeutics:

  • The highly specific expression pattern of ANTXR2 in peripheral sensory neurons with minimal CNS expression presents a unique opportunity for developing peripherally restricted analgesics .

  • Such therapeutics could potentially avoid central nervous system side effects that limit many current pain medications.

  • ANTXR2 antibodies are essential for validating target engagement in preclinical models.

• Engineered Molecular Delivery Systems:

  • Modified anthrax toxin components can be engineered to deliver therapeutic cargo specifically to ANTXR2-expressing neurons .

  • This approach could enable targeted delivery of analgesic molecules, gene therapy vectors, or silencing RNAs to pain-sensing neurons.

  • Immunohistochemical validation with ANTXR2 antibodies confirms the specificity of these delivery systems.

• Therapeutic Potential in Different Pain Conditions:

  • Current research suggests potential efficacy in:

    • Inflammatory pain models

    • Neuropathic pain states

    • Post-surgical pain

  • Antibody-based detection of ANTXR2 in human DRG samples can help identify patient populations most likely to benefit from ANTXR2-targeted therapies.

This emerging field represents a paradigm shift in our understanding of ANTXR2 function beyond its role as an anthrax toxin receptor, highlighting its significance in pain neurobiology and presenting novel opportunities for analgesic drug development. Methodological approaches using ANTXR2 antibodies will continue to be essential for advancing this research frontier.

How is ANTXR2 research contributing to our understanding of rare genetic disorders?

ANTXR2 mutations are causally linked to a spectrum of rare genetic disorders, primarily infantile systemic hyalinosis (ISH) and juvenile hyaline fibromatosis (JHF), collectively termed hyaline fibromatosis syndrome (HFS). Advanced research utilizing ANTXR2 antibodies has significantly enhanced our understanding of these disorders through multiple investigative approaches:

Molecular Characterization of ANTXR2 Mutations in Hyaline Fibromatosis Syndrome:

• Mutation-Function Correlation Studies:

  • ANTXR2 antibodies enable assessment of protein expression, localization, and function in patient-derived samples.

  • Western blot analysis using specific antibodies can determine if mutations result in:

    • Reduced protein expression

    • Truncated protein products

    • Altered post-translational modifications

  • Comparative immunohistochemistry between normal and patient tissues reveals altered distribution patterns and expression levels .

• Domain-Specific Impact Assessment:

  • Many HFS-causing mutations affect the von Willebrand factor A (vWA) domain containing the metal ion-dependent adhesion site (MIDAS) motif .

  • These mutations have been shown to inhibit binding by the vWA domain to its ligands.

  • Structural and functional analyses using domain-specific ANTXR2 antibodies help map the consequences of specific mutations.

  • Immunoprecipitation studies can reveal disrupted protein-protein interactions caused by mutations.

• Genotype-Phenotype Correlations:

  • Severity of clinical manifestations correlates with the functional impact of specific ANTXR2 mutations.

  • Complete loss-of-function mutations typically result in the more severe infantile systemic hyalinosis.

  • Partial function-preserving mutations often lead to the milder juvenile hyaline fibromatosis.

  • Immunohistochemical quantification using ANTXR2 antibodies helps establish relationships between protein expression/localization and clinical severity.

Pathophysiological Mechanisms in ANTXR2-Related Disorders:

• Disrupted Extracellular Matrix Interactions:

  • ANTXR2 normally binds to basement membrane components including laminin and collagen type IV .

  • Mutations disrupt these interactions, leading to abnormal extracellular matrix deposition.

  • Co-immunoprecipitation studies with ANTXR2 antibodies can identify altered binding to matrix proteins.

  • Immunofluorescence co-localization can visualize disrupted matrix organization in patient samples.

• Altered Cellular Signaling Pathways:

  • ANTXR2 mutations may affect downstream signaling pathways.

  • Phosphorylation state analysis of signaling molecules in wild-type versus mutant cells.

  • Antibody-based proteomics approaches can identify dysregulated pathways.

  • These studies help establish whether HFS results from loss-of-function or gain-of-function mechanisms.

• Impact on Angiogenesis and Vascular Function:

  • Given ANTXR2's role in capillary morphogenesis, mutations may disrupt normal vascular development .

  • Immunohistochemical analysis of blood vessel density and morphology in patient tissues.

  • In vitro angiogenesis assays comparing wild-type and mutant ANTXR2 function.

  • These studies help explain the vascular anomalies observed in some HFS patients.

Experimental Models for ANTXR2-Related Disorders:

• Patient-Derived Cell Models:

  • Fibroblasts or induced pluripotent stem cells (iPSCs) from patients with ANTXR2 mutations.

  • ANTXR2 antibodies can confirm mutation effects on protein expression and localization.

  • These cell models allow for detailed biochemical and functional studies.

  • Drug screening platforms to identify compounds that rescue mutant ANTXR2 function.

• Genetic Animal Models:

  • Knockin mice harboring specific HFS-causing ANTXR2 mutations.

  • Conditional knockout models to study tissue-specific effects.

  • Immunohistochemical analysis using ANTXR2 antibodies to validate model fidelity.

  • Temporal expression studies to understand developmental aspects of the disorder.

• In Vitro 3D Tissue Models:

  • Organoid cultures from patient-derived cells.

  • Assessment of ANTXR2 expression and function in complex tissue environments.

  • These models bridge the gap between simple cell culture and animal models.

  • Antibody-based imaging of 3D structures reveals organizational defects caused by mutations.

Diagnostic and Therapeutic Developments:

• Improved Diagnostic Approaches:

  • ANTXR2 antibodies enable immunohistochemical diagnosis from skin or gingival biopsies.

  • Standardized protocols for evaluating ANTXR2 expression and localization patterns.

  • Complementary approach to genetic testing for confirming pathogenicity of novel variants.

  • Particularly valuable in regions where genetic testing is not readily available.

• Therapeutic Strategy Development:

  • Small molecule screening to identify compounds that can rescue mutant ANTXR2 trafficking or function.

  • Chaperone therapies to stabilize mutant proteins.

  • Gene therapy approaches to replace defective ANTXR2.

  • ANTXR2 antibodies are essential for validating therapeutic efficacy by assessing proper protein expression and localization.

• Enzyme Replacement and Protein Supplementation Approaches:

  • Recombinant soluble ANTXR2 ectodomains as potential replacement therapy.

  • Engineering of modified ANTXR2 proteins that can compensate for mutant function.

  • Antibody-based detection methods to monitor biodistribution and tissue penetration of therapeutic proteins.

Beyond Hyaline Fibromatosis: Expanded Understanding of ANTXR2 in Other Disorders:

• ANTXR2 in Cancer Biology:

  • Immunohistochemical profiling of ANTXR2 expression across different tumor types.

  • Correlation with tumor angiogenesis, invasion, and metastasis.

  • Potential role as a prognostic biomarker or therapeutic target.

  • These studies broaden our understanding of ANTXR2 function in pathological processes beyond rare genetic disorders.

• ANTXR2 in Fibrotic Disorders:

  • Assessment of ANTXR2 expression in other fibrotic conditions beyond HFS.

  • Investigation of common pathways between HFS and other fibrotic diseases.

  • Potential therapeutic implications for more common fibrotic disorders.

  • Antibody-based tissue profiling is essential for establishing these connections.

• ANTXR2 Polymorphisms and Disease Susceptibility:

  • Investigation of how common ANTXR2 genetic variations affect protein function and disease risk.

  • Assessment of SNPs that influence ANTXR2 expression and their association with various conditions.

  • Studies have demonstrated correlations between specific SNP combinations and anthrax toxin sensitivity .

  • These findings may have implications for understanding variable disease susceptibility or therapeutic responses.

The investigation of ANTXR2's role in rare genetic disorders has not only advanced our understanding of these specific conditions but has also provided broader insights into fundamental biological processes related to extracellular matrix organization, angiogenesis, and cell-matrix interactions. ANTXR2 antibodies continue to serve as indispensable tools in this evolving field of research.