EXPH5 Antibody

What is EXPH5 and what function does it serve in cellular processes?

Exophilin 5 (EXPH5, also known as SLAC2B or KIAA0624) functions as a Rab effector protein primarily involved in vesicle trafficking pathways . It belongs to the exophilin family of proteins that mediate interactions between vesicles and the cytoskeleton. Recent studies using knockout models have revealed that EXPH5 plays significant roles in regulating extracellular superoxide release, intracellular reactive oxygen species (ROS) production, and phosphoinositide 3-kinase activity . This regulation occurs through its control over intracellular trafficking mechanisms. Structurally, EXPH5 contains domains that facilitate interaction with both membrane components and cytoskeletal elements, allowing it to serve as a molecular bridge in vesicular transport processes.

What are the primary applications of EXPH5 antibodies in research methodologies?

EXPH5 antibodies are predominantly employed in several key methodologies:

• Immunohistochemistry (IHC): Used for detecting and localizing EXPH5 in tissue sections, providing insights into spatial distribution within organs and cellular compartments .

• Western Blotting (WB): Applied for quantifying EXPH5 protein levels and confirming protein size in various experimental conditions.

• ELISA: Utilized for quantitative detection of EXPH5 in solution-based assays .

• Immunofluorescence: When conjugated with fluorophores like FITC, these antibodies enable visualization of EXPH5 localization in fixed cells and tissues .

The selection of specific methodology depends on research objectives, with considerations for sensitivity, specificity, and whether qualitative or quantitative data is required.

How should researchers select appropriate EXPH5 antibodies for specific applications?

Selection of appropriate EXPH5 antibodies requires consideration of multiple experimental parameters:

• Target epitope relevance: Antibodies targeting different amino acid regions (e.g., AA 1235-1490 vs. AA 1907-1956) may yield different results depending on protein conformation and experimental conditions .

• Host species and clonality: While rabbit polyclonal antibodies offer broader epitope recognition, this must be balanced against experimental requirements for specificity.

• Conjugation requirements: Consider whether unconjugated antibodies are sufficient or if conjugated versions (HRP, FITC, biotin) are necessary for detection methods .

• Cross-reactivity profile: Validate that the antibody's reactivity pattern across species (human, cow, dog, rat, etc.) aligns with experimental models .

• Validation data: Assess whether the antibody has been validated in applications similar to your planned experiments, particularly for challenging techniques like immunohistochemistry.

What are the optimal storage and handling procedures for maintaining EXPH5 antibody efficacy?

To maintain optimal EXPH5 antibody performance, adhere to these evidence-based handling protocols:

• Storage temperature: Store antibodies at -20°C or -80°C for long-term preservation. Repeated freeze-thaw cycles significantly compromise antibody functionality .

• Buffer composition: EXPH5 antibodies are typically preserved in buffers containing 50% glycerol and 0.01M PBS (pH 7.4) with 0.03% Proclin 300 as a preservative . This composition helps maintain protein stability during storage.

• Aliquoting strategy: Upon receipt, divide antibodies into single-use aliquots to minimize freeze-thaw cycles and potential contamination.

• Thawing protocols: Thaw antibodies completely at 4°C before use, avoid room temperature thawing which can accelerate degradation.

• Handling precautions: Some preparations contain ProClin, which is classified as hazardous and should be handled only by trained personnel using appropriate safety measures .

How does EXPH5 relate to genetic disorders, particularly epidermolysis bullosa?

Research has identified significant connections between EXPH5 mutations and epidermolysis bullosa (EB), a heterogeneous group of heritable blistering diseases. Next-generation sequencing has revealed homozygous mutations in EXPH5 that contribute to EB pathogenesis .

The specific mutation chr11 g.108510085G>A (p.Arg1808Ter) in EXPH5 has been documented in a patient with a complex EB phenotype who simultaneously carried a COL17A1 mutation . Transmission electron microscopy and immunostaining confirmed features consistent with both EB simplex and junctional EB subtypes. This case highlights the potential for digenic inheritance patterns in EB, where mutations in two distinct genes contribute to a complex clinical presentation.

For researchers investigating genetic skin disorders, EXPH5 should be included in mutational analysis panels, particularly in cases with atypical phenotypic presentations or consanguineous family backgrounds. The identification of such mutations has significant implications for genetic counseling and potential prenatal testing strategies .

What role does EXPH5 play in immunological research, particularly in allergic airway inflammation models?

Exophilin-5 demonstrates significant regulatory functions in allergic airway inflammation through several mechanisms:

• Cytokine regulation: Studies using Exph5-knockout mice have shown that exophilin-5 deficiency leads to enhanced production of Th2 cytokines (particularly IL-4, IL-5, and IL-13) without affecting IFN-γ (a Th1 cytokine) . This suggests a specific regulatory role in Th2-mediated immune responses.

• Eosinophilic inflammation: Exph5-KO mice exhibit markedly enhanced eosinophil infiltration in the lungs in an OVA-induced asthma model .

• Mucus production: Exophilin-5 deficiency results in increased mucus production in the lungs during allergic airway inflammation .

The experimental data from both in vivo models and ex vivo cell culture systems indicates that exophilin-5 functions as a negative regulator of Th2-mediated allergic responses. This regulatory role appears to involve control of cellular trafficking mechanisms that influence cytokine production and release.

What methodological considerations are essential when using EXPH5 antibodies in investigative immunohistochemistry?

When employing EXPH5 antibodies for immunohistochemistry, researchers should implement these methodological approaches for optimal results:

How can EXPH5 antibodies be integrated with advanced genetic analysis techniques?

Integration of EXPH5 antibody-based detection methods with genetic analysis techniques enables multilevel characterization of EXPH5 function and pathology:

• Correlation with next-generation sequencing (NGS) data: As demonstrated in EB research, combining immunohistochemical staining patterns using EXPH5 antibodies with NGS mutational analysis provides comprehensive genotype-phenotype correlations . This approach has successfully identified cases where genetic mutations lead to truncated proteins or altered expression patterns.

• RNAseq complementation: In cases where EXPH5 mutations are identified, RNAseq analysis can reveal complex splicing patterns, as demonstrated in COL17A1 mutations . Similar approaches can be applied to EXPH5 mutations to understand their impact on transcript processing.

• Chromatin immunoprecipitation (ChIP) applications: EXPH5 antibodies can be utilized in ChIP experiments to investigate epigenetic regulation of EXPH5 expression or to study potential transcription factor binding that may influence EXPH5 levels.

• Single-cell analysis integration: Combining immunofluorescence using EXPH5 antibodies with single-cell transcriptomics creates powerful approaches for understanding cell-type specific expression patterns and functional variations.

• CRISPR-Cas9 validation: EXPH5 antibodies provide essential validation tools for CRISPR-Cas9 modified cell lines, confirming successful gene editing at the protein level.

What are the emerging applications of EXPH5 research in therapeutic development?

While EXPH5 itself is not currently a direct therapeutic target, research into its functions informs several potential therapeutic development avenues:

• Allergic airway inflammation therapeutics: The role of exophilin-5 in regulating Th2 cytokine production and eosinophilic inflammation suggests potential applications in asthma and allergic disease management . Understanding this regulatory pathway could inform development of novel targeted therapies.

• Genetic skin disorder treatments: The identification of EXPH5 mutations in epidermolysis bullosa patients opens avenues for gene therapy approaches or protein replacement strategies . This research contributes to the broader understanding of molecular mechanisms underlying EB.

• Vesicular trafficking modulators: As a Rab effector protein involved in vesicle trafficking, EXPH5 research informs development of therapeutics targeting cellular transport mechanisms . Such approaches could potentially address disorders characterized by defective protein trafficking.

• Bispecific antibody development: The methodologies used in studying EXPH5 contribute to broader antibody engineering approaches. Recent advances in bispecific and multivalent antibody development, including co-targeting strategies and tetravalent constructs, may be applicable to EXPH5-related pathway modulation .

What are common technical challenges when working with EXPH5 antibodies and how can they be addressed?

Researchers frequently encounter these technical challenges when working with EXPH5 antibodies:

• Background staining in immunohistochemistry:

  • Challenge: Non-specific binding resulting in high background noise

  • Solution: Implement more stringent blocking protocols using 5-10% normal serum from the same species as the secondary antibody; consider adding 0.1-0.3% Triton X-100 for improved permeabilization while reducing non-specific binding

• Inconsistent western blot results:

  • Challenge: Variable detection of EXPH5 protein bands

  • Solution: Optimize lysis buffer composition to ensure complete solubilization of membrane-associated proteins; add protease inhibitors immediately before use; consider gradient gels for better resolution of high molecular weight proteins

• Epitope masking in fixed tissues:

  • Challenge: Formaldehyde fixation can mask epitopes recognized by EXPH5 antibodies

  • Solution: Evaluate multiple antigen retrieval methods systematically, comparing heat-induced epitope retrieval with enzymatic retrieval approaches

• Cross-reactivity with related proteins:

  • Challenge: Potential cross-reactivity with other exophilin family members

  • Solution: Validate antibody specificity using knockout controls or competitive binding assays with recombinant proteins

• Low signal strength in low-expressing tissues:

  • Challenge: Insufficient detection in tissues with naturally low EXPH5 expression

  • Solution: Implement signal amplification systems such as tyramide signal amplification; consider longer primary antibody incubation at 4°C (overnight to 48 hours)

How does EXPH5 expression pattern vary across different tissue types and experimental models?

EXPH5 demonstrates distinctive expression patterns across tissues and experimental models that researchers should consider when designing studies:

• Immune cell expression:

  • Documented expression in human CD4+ T cells, particularly in Th2-enriched CD4+ cell fractions

  • Presence in the human CD4+ T cell line Jurkat

  • Differential expression patterns between activated and resting lymphocytes

• Skin tissue expression:

  • Relevant expression in skin tissues, particularly significant in the context of epidermolysis bullosa research

  • Localization patterns that may vary between epidermal layers

• Respiratory system presence:

  • Functional significance in lung tissue related to allergic airway inflammation models

  • Expression levels that may be altered in inflammatory conditions

• Model-specific considerations:

  • Exph5-knockout mice exhibit normal development without gross phenotypic abnormalities in body weight, growth, organ structures, or immune cell composition in peripheral lymph tissues

  • These knockout models do not spontaneously develop eosinophilic lung inflammation, suggesting compensatory mechanisms or requirement for specific triggers

• Species variations:

  • Available antibodies show reactivity across multiple species including human, cow, dog, horse, rabbit, and rat, indicating evolutionary conservation

  • Species-specific isoforms may exist and should be considered when selecting appropriate antibody reagents

What are promising future research avenues involving EXPH5 and related molecular mechanisms?

Several promising research directions can expand our understanding of EXPH5 biology and potential applications:

• Comprehensive characterization of EXPH5 interactome:

  • Identify protein-protein interactions involving EXPH5 to better understand its role in vesicular trafficking

  • Map binding partners under different cellular conditions (resting, activated, stressed)

  • Develop interaction network maps to position EXPH5 within broader cellular pathways

• EXPH5 roles in additional immune contexts:

  • Explore functions beyond Th2 responses, examining potential roles in other T cell subsets

  • Investigate involvement in innate immune responses

  • Examine potential contributions to autoimmune disorders beyond allergic conditions

• Structural biology approaches:

  • Determine high-resolution structures of EXPH5 domains

  • Characterize conformational changes associated with binding to Rab proteins

  • Develop structure-based therapeutic design targeting EXPH5-dependent pathways

• Expanded genetic association studies:

  • Screen for EXPH5 mutations in additional dermatological disorders beyond epidermolysis bullosa

  • Conduct genome-wide association studies to identify conditions where EXPH5 variants contribute to pathology

  • Evaluate potential roles as a disease modifier gene in complex disorders

• Advanced tissue-specific knockout models:

  • Develop conditional knockout models to evaluate tissue-specific functions

  • Generate knock-in models of disease-associated mutations

  • Implement time-controlled gene manipulation to assess developmental versus maintenance roles

How can researchers effectively validate novel findings related to EXPH5 function?

Rigorous validation of novel EXPH5 findings requires multilevel approaches:

• Complementary methodological validation:

  • Combine antibody-based detection with genetic approaches (RNA interference, CRISPR-Cas9)

  • Validate findings across multiple cell types or experimental models

  • Implement rescue experiments where gene function is restored after knockout/knockdown

• Cross-platform confirmation:

  • Validate protein-level findings with transcript analysis

  • Confirm in vitro results in appropriate in vivo models

  • Translate animal model findings to human samples when possible

• Independent antibody validation:

  • Use multiple antibodies targeting different epitopes of EXPH5

  • Implement epitope-tagged overexpression systems as complementary approaches

  • Include appropriate controls for antibody specificity in each experimental system

• Functional consequence assessment:

  • Measure downstream effects of EXPH5 manipulation on relevant physiological processes

  • Quantify phenotypic changes using standardized assays

  • Document dose-response relationships where applicable

• Reproducibility considerations:

  • Implement blinded analysis for subjective measurements

  • Pre-register experimental designs and analysis plans

  • Share detailed methodological protocols to facilitate independent replication

Data Tables and Research Resources

Effect of EXPH5 Knockout on Cytokine Production in Allergic Airway Inflammation

What is the recommended protocol for EXPH5 immunohistochemical staining?

The following protocol represents a methodological foundation for EXPH5 immunohistochemical staining, which should be optimized for specific research contexts:

• Tissue preparation:

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

  • Process and embed in paraffin following standard histological procedures

  • Section tissues at 4-5μm thickness onto positively charged slides

• Deparaffinization and rehydration:

  • Deparaffinize sections in xylene (3 × 5 minutes)

  • Rehydrate through graded alcohols (100%, 95%, 70%) to distilled water

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • Bring to boil and maintain at sub-boiling temperature for 20 minutes

  • Allow to cool at room temperature for 20 minutes

• Blocking steps:

  • Block endogenous peroxidase activity with 3% hydrogen peroxide for 10 minutes

  • Block non-specific binding with 5% normal goat serum in PBS for 1 hour at room temperature

• Primary antibody incubation:

  • Dilute EXPH5 antibody (AA 1235-1490) in antibody diluent at 1:20-1:200 (optimal dilution should be determined empirically)

  • Incubate overnight at 4°C in a humidified chamber

• Secondary antibody and detection:

  • Apply appropriate HRP-conjugated secondary antibody for 30-60 minutes at room temperature

  • Develop with DAB substrate for 5-10 minutes (monitor microscopically)

  • Counterstain with hematoxylin for 1-2 minutes

• Dehydration and mounting:

  • Dehydrate through graded alcohols to xylene

  • Mount with permanent mounting medium

• Controls to include:

  • Positive control tissue (human T cell-rich tissue)

  • Negative control (primary antibody omission)

  • Isotype control (non-specific IgG at equivalent concentration)