HOXC10 Antibody

What is HOXC10 and why is it important for research studies?

HOXC10 (Homeobox C10) is a transcription factor belonging to the Abd-B homeobox protein family. In humans, canonical HOXC10 protein has 342 amino acid residues with a mass of 38.1 kDa, though it often appears around 50 kDa in Western blots . HOXC10 plays critical roles in:

• Regulation of gene expression during development

• Cellular differentiation and morphogenesis

• Patterning along the anterior-posterior axis

• Cell proliferation and cell cycle control

HOXC10 has become increasingly important in research due to its involvement in various pathological conditions, including:

• Cancer progression and chemotherapy resistance in breast cancer

• Immunosuppression in glioma

• Metastasis in colorectal and ovarian cancers

• Metabolic regulation in adipose tissue

What applications are most effective for HOXC10 antibody detection?

HOXC10 antibodies are validated for multiple applications, with varying effectiveness depending on the experimental context:

Western blot is particularly effective for quantitative analysis, while IHC and IF provide valuable spatial information about HOXC10 localization within tissues and cells . For detecting protein-DNA interactions, chromatin immunoprecipitation (ChIP) has proven effective for identifying HOXC10 binding to regulatory regions of target genes .

How do I optimize protein extraction to preserve HOXC10 integrity for antibody detection?

HOXC10 is primarily localized in the nucleus, making proper nuclear extraction critical for successful detection. A methodological approach includes:

• Tissue/Cell Preparation: Fresh or flash-frozen samples yield better results than long-stored samples

• Nuclear Extraction Protocol:

  • Use specialized nuclear extraction buffers containing protease inhibitors

  • Include phosphatase inhibitors if studying phosphorylation states

  • Perform gentle mechanical disruption followed by detergent-based lysis

  • Separate cytoplasmic and nuclear fractions by centrifugation

• Protein Denaturation: HOXC10 may require stronger denaturation conditions (higher SDS, boiling) for complete epitope exposure

• Storage Considerations: Aliquot extracts and store at -80°C to avoid freeze-thaw cycles that can degrade HOXC10

Research has shown that HOXC10 protein levels can be affected by proteasomal degradation, particularly during cold exposure in adipose tissue . Therefore, including proteasome inhibitors in extraction buffers may be necessary when studying regulated degradation of HOXC10.

How can I validate HOXC10 antibody specificity for my experimental systems?

Rigorous validation is essential for ensuring HOXC10 antibody specificity:

• Positive and Negative Controls:

  • Positive controls: A549 cells, HepG2 cells, and mouse heart tissue have been validated for HOXC10 expression

  • Negative controls: Use HOXC10 knockdown/knockout cells or tissues

  • Overexpression systems: Ectopic expression of tagged HOXC10 provides additional validation

• Cross-Reactivity Assessment:

  • Test in multiple species if working across evolutionary boundaries

  • Check for potential cross-reactivity with other HOX family members, particularly HOXC6, HOXC8, and HOXC13 due to sequence similarity

• Multiple Antibody Validation:

  • Use antibodies targeting different epitopes of HOXC10

  • Compare monoclonal and polyclonal antibodies

  • Validate with different detection methods (fluorescence vs. chromogenic)

• Molecular Weight Verification:

  • Human HOXC10 has a calculated molecular weight of 38.1 kDa but is often detected at approximately 50 kDa in Western blots due to post-translational modifications

  • Multiple bands may indicate isoforms, degradation products, or non-specific binding

Research by Pathak et al. used siRNA-mediated knockdown of HOXC10 in ovarian cancer cell lines to validate antibody specificity, showing significantly reduced signal in Western blots of knockdown cells compared to control cells .

What are the optimal protocols for HOXC10 chromatin immunoprecipitation (ChIP) assays?

ChIP assays for HOXC10 require specific optimization due to its role as a transcription factor:

• Chromatin Preparation:

  • Crosslink with 1% formaldehyde for 10 minutes at room temperature

  • Quench with 125mM glycine

  • Sonicate to achieve chromatin fragments of 200-500bp

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Use 2-5μg of validated ChIP-grade HOXC10 antibody per reaction

  • Include IgG control and input samples

  • Incubate overnight at 4°C with rotation

• Washing and Elution:

  • Use increasingly stringent wash buffers to reduce background

  • Elute at 65°C in elution buffer containing SDS

  • Reverse crosslinks overnight at 65°C

• Target Analysis:

  • Analyze by qPCR with primers designed for known or predicted HOXC10 binding sites

  • Consider ChIP-seq for genome-wide binding profile

Multiple studies have successfully employed ChIP to demonstrate HOXC10 binding to promoter regions of target genes. For example, Pathak et al. performed ChIP-PCR and ChIP-qPCR to show HOXC10 binding to the CST1 promoter region (Chr20:23,732,257-23,732,507) in gastric cancer cells . Similarly, another study revealed HOXC10 binding to the -1667 to -1412 region of the PRDM16 5' regulatory region in adipose tissue .

How can I effectively use HOXC10 antibodies for multiplex imaging in complex tissues?

Multiplex imaging with HOXC10 antibodies requires careful optimization:

• Antibody Selection:

  • Choose antibodies raised in different host species for co-staining

  • Ensure minimal cross-reactivity between detection systems

  • Validate each antibody individually before multiplexing

• Tissue Preparation:

  • Optimize fixation protocols (4% PFA for 24h is standard)

  • Consider antigen retrieval methods (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

  • Test blocking solutions to minimize background

• Detection Strategy:

  • Sequential immunostaining may be necessary for same-species antibodies

  • Use tyramide signal amplification for weak signals

  • Consider spectral unmixing for fluorophores with overlapping emission spectra

• Controls for Multiplex Imaging:

  • Single-stain controls for each antibody

  • Secondary-only controls

  • HOXC10 knockdown tissue as negative control

Research has employed multiplex imaging to study HOXC10 in relation to other markers in breast cancer tissues, revealing associations between HOXC10 expression and specific cancer subtypes, particularly in relation to chemotherapy resistance mechanisms .

Why might I be observing inconsistent HOXC10 detection in Western blots?

Inconsistent HOXC10 detection can result from several factors:

• Protein Degradation:

  • HOXC10 undergoes proteasome-mediated degradation, particularly in response to cAMP-dependent protein kinase activation

  • Solution: Include fresh protease inhibitors and proteasome inhibitors (MG132) in extraction buffers

• Inefficient Nuclear Extraction:

  • As a nuclear protein, HOXC10 requires efficient nuclear extraction

  • Solution: Optimize nuclear extraction protocol with appropriate detergents and mechanical disruption

• Antibody Specificity Issues:

  • Some antibodies may detect specific HOXC10 post-translational modifications

  • Solution: Test multiple antibodies targeting different epitopes

• Sample-Specific Variability:

  • HOXC10 expression varies significantly between tissues and cell types

  • Solution: Include positive controls (A549 cells, mouse heart tissue) alongside experimental samples

• Loading Control Discrepancies:

  • Nuclear loading controls may not reflect HOXC10 levels accurately

  • Solution: Use nuclear-specific loading controls like Lamin B1 or histone H3

Studies have shown that HOXC10 protein levels can fluctuate based on physiological conditions, such as cold exposure in adipose tissue, which triggers its degradation via the proteasome pathway . This degradation is mediated by E3 ligases KCTD2, 5, and 17, which should be considered when analyzing HOXC10 levels in different experimental conditions.

How can I improve signal-to-noise ratio when using HOXC10 antibodies for immunohistochemistry?

Improving signal-to-noise ratio for HOXC10 IHC requires systematic optimization:

• Tissue Fixation and Processing:

  • Optimize fixation time (overfixation can mask epitopes)

  • Use freshly prepared fixatives

  • Ensure consistent tissue processing conditions

• Antigen Retrieval:

  • Test both heat-induced epitope retrieval (HIER) and enzymatic retrieval

  • For HIER, compare citrate buffer (pH 6.0) vs. EDTA buffer (pH 9.0)

  • Optimize retrieval time and temperature

• Blocking Optimization:

  • Use species-specific serum matching secondary antibody

  • Add 0.1-0.3% Triton X-100 for improved antibody penetration

  • Consider adding avidin/biotin blocking for biotin-based detection systems

• Antibody Incubation:

  • Test various antibody dilutions (typically 1:50-1:500)

  • Optimize incubation temperature and time (4°C overnight often yields better results than room temperature incubation)

  • Use antibody diluent with background-reducing components

• Signal Development:

  • For chromogenic detection, optimize DAB development time

  • For fluorescence, use TSA amplification for weak signals

  • Include DAPI counterstain to visualize nuclei for proper HOXC10 localization

Research has successfully employed HOXC10 immunohistochemistry to evaluate expression in colorectal cancer tissues, showing significantly higher expression in tumor tissues compared to non-tumor tissues and correlating with clinical parameters like tumor differentiation, invasion, and metastasis .

What experimental controls are essential when studying HOXC10 knockdown or overexpression?

Rigorous controls are critical for HOXC10 genetic manipulation studies:

• Knockdown Controls:

  • Multiple siRNA sequences targeting different regions of HOXC10 mRNA to rule out off-target effects

  • Non-targeting siRNA with similar GC content as negative control

  • Measurement of knockdown efficiency at both mRNA (qRT-PCR) and protein (Western blot) levels

  • Rescue experiments with siRNA-resistant HOXC10 cDNA

• Overexpression Controls:

  • Empty vector controls processed identically to HOXC10-expressing vectors

  • Tagged HOXC10 constructs (FLAG, HA) for distinguishing endogenous from exogenous protein

  • Verification of nuclear localization of overexpressed HOXC10

  • Mutant HOXC10 controls (e.g., DNA-binding mutants) to confirm specificity

• Functional Validation:

  • Measurement of known HOXC10 target genes (e.g., PRDM16, CST1)

  • Phenotypic assays relevant to HOXC10 function (e.g., cell proliferation, migration, thermogenesis)

• Cell Type Considerations:

  • Cell lines with low endogenous HOXC10 for overexpression studies (AGS, MKN74)

  • Cell lines with high endogenous HOXC10 for knockdown studies (SNU-216, SNU-484)

Studies investigating HOXC10 function in gastric cancer demonstrated effective knockdown validation by using RT-qPCR and Western blot analysis to confirm reduced expression at both mRNA and protein levels. Similarly, overexpression was confirmed using both techniques before proceeding with functional assays measuring proliferation, colony formation, and migration .

How is HOXC10 expression correlated with patient outcomes in cancer research?

Multiple studies have established significant correlations between HOXC10 expression and patient outcomes across various cancers:

These correlations suggest HOXC10 as a potential prognostic biomarker and therapeutic target across multiple cancer types.

What methodological approaches are used to study HOXC10's role in chemotherapy resistance?

Investigating HOXC10's contribution to chemotherapy resistance employs several specialized techniques:

• Cell Viability and Cytotoxicity Assays:

  • MTT/MTS assays comparing survival of HOXC10-overexpressing vs. control cells after drug treatment

  • Colony formation assays to assess long-term survival after chemotherapy exposure

  • Flow cytometry with Annexin V/PI staining to quantify apoptotic populations

• DNA Damage Response Assessment:

  • Immunofluorescence for γH2AX foci to measure DNA double-strand breaks

  • Comet assay to directly visualize DNA damage

  • Western blot analysis of DNA damage response proteins (ATM, ATR, BRCA1/2)

• Homologous Recombination Measurement:

  • HR reporter assays using DR-GFP constructs

  • Immunofluorescence for RAD51 foci formation

  • ChIP assays to detect HOXC10 recruitment to DNA damage sites

• Cell Cycle Analysis:

  • Flow cytometry with propidium iodide for cell cycle distribution

  • Western blot for cyclins and CDKs

  • BrdU incorporation to measure S-phase progression

• In Vivo Models:

  • Patient-derived xenografts with manipulated HOXC10 expression

  • Treatment with clinically relevant chemotherapy regimens

  • Assessment of tumor response, recurrence, and metastasis

Research has revealed that HOXC10 enhances S-phase-specific DNA damage repair by homologous recombination and checkpoint recovery through recruitment of HR proteins to DNA damage sites. HOXC10 facilitates these processes by binding to and activating cyclin-dependent kinase CDK7, which regulates transcription by phosphorylating RNA polymerase II .

How can HOXC10 antibodies be used to investigate the role of HOXC10 in adipose tissue metabolism?

HOXC10 antibodies are valuable tools for studying adipose tissue metabolism:

• Tissue-Specific Expression Analysis:

  • Western blot analysis of HOXC10 in different adipose depots (subcutaneous vs. visceral)

  • Immunohistochemistry to visualize HOXC10 expression patterns within adipose tissue

  • Flow cytometry of stromal vascular fraction vs. mature adipocytes

• Cold Adaptation Studies:

  • Time-course analysis of HOXC10 protein levels during cold exposure

  • Co-immunoprecipitation to identify interacting partners during thermal challenges

  • Detection of post-translational modifications associated with HOXC10 degradation

• Chromatin Analysis:

  • ChIP-seq to identify genome-wide HOXC10 binding sites in adipocytes

  • ChIP-qPCR for specific targets like PRDM16 promoter

  • Co-ChIP to study HOXC10 interaction with other transcription factors

• Protein Degradation Monitoring:

  • Immunoprecipitation of ubiquitinated HOXC10

  • Pulse-chase experiments to measure HOXC10 half-life

  • Co-IP with E3 ligases like KCTD2, 5, and 17

Research has established that HOXC10 is a critical suppressor of browning in white adipose tissue. HOXC10 knockout mice exhibit spontaneous browning of subcutaneous WAT with increased expression of brown-fat markers (Prdm16, Pgc-1α, Ucp1, Dio2, and CideA), higher basal body temperature, protection against hypothermia during cold challenge, and improved glucose tolerance and insulin sensitivity .

What experimental approaches best demonstrate HOXC10's transcriptional regulatory function?

As a transcription factor, HOXC10's regulatory function can be investigated through multiple complementary approaches:

• Gene Expression Analysis:

  • RNA-sequencing of cells with modulated HOXC10 expression

  • qRT-PCR validation of identified target genes

  • Pathway enrichment analysis of differentially expressed genes

• DNA Binding Characterization:

  • ChIP-seq to identify genome-wide binding sites

  • Motif analysis to determine HOXC10 binding preferences

  • EMSAs to validate direct DNA binding to specific sequences

• Reporter Assays:

  • Luciferase reporters containing promoters of putative target genes

  • Mutation analysis of HOXC10 binding sites

  • Dose-response studies with varying HOXC10 expression levels

• Protein-Protein Interactions:

  • Co-immunoprecipitation to identify transcriptional cofactors

  • Proximity ligation assays to visualize interactions in situ

  • Mass spectrometry to identify HOXC10-associated protein complexes

• Functional Validation:

  • CRISPR-mediated deletion of HOXC10 binding sites

  • Rescue experiments with wild-type vs. DNA-binding mutants

  • Simultaneous knockdown/overexpression of HOXC10 and target genes

Research has demonstrated HOXC10's direct transcriptional regulation of multiple genes. For example, in glioma cells, HOXC10 directly binds to the promoter regions of PD-L2 and TDO2, regulating immunosuppressive gene expression . In adipose tissue, HOXC10 suppresses thermogenesis by binding to the 5' regulatory region of PRDM16 (-1667 to -1412), as confirmed by chromatin immunoprecipitation and mobility shift assays .

How do experimental conditions affect HOXC10 protein stability and detection?

HOXC10 protein stability is highly sensitive to experimental conditions:

• Temperature Effects:

  • Cold exposure (4°C) triggers proteasome-mediated degradation of HOXC10 in adipocytes

  • This degradation is cAMP-dependent protein kinase (PKA)-dependent

  • E3 ligases KCTD2, 5, and 17 have been identified as regulators of HOXC10 stability

• Cell Cycle Regulation:

  • HOXC10 levels oscillate throughout the cell cycle

  • The protein is targeted for degradation early in mitosis via the ubiquitin-dependent proteasome pathway

  • Cell synchronization may be necessary for consistent detection

• Fixation and Extraction Conditions:

  • Paraformaldehyde fixation may mask certain HOXC10 epitopes

  • Methanol fixation may better preserve nuclear antigens

  • Nuclear extraction protocols significantly impact detection efficiency

• Post-translational Modifications:

  • Phosphorylation states can affect antibody recognition

  • Ubiquitination changes protein mobility in SDS-PAGE

  • Proteasome inhibitors (MG132) may reveal otherwise rapidly degraded forms

Research has shown that in adipose tissue, cold exposure induces the degradation of HOXC10 protein without affecting mRNA levels, suggesting post-translational regulation . This degradation mechanism represents an important regulatory pathway controlling HOXC10 function in metabolic adaptation.

What are the most informative experimental models for studying HOXC10 function across different tissue types?

Different research questions require specific experimental models:

• Cancer Biology:

  • Cell lines: AGS and MKN74 (low HOXC10) for overexpression; SNU-216 and SNU-484 (high HOXC10) for knockdown studies in gastric cancer

  • Orthotopic models: Cecum injection for colorectal cancer metastasis

  • Patient-derived xenografts: For chemotherapy resistance studies in breast cancer

• Adipose Tissue Metabolism:

  • Adipocyte-specific HOXC10 knockout mice (via Adiponectin-Cre)

  • AAV8-mediated HOXC10 overexpression in adipose tissue

  • Primary adipocyte cultures from different depots

  • 3T3-L1 pre-adipocyte differentiation system

• Developmental Biology:

  • Embryonic stem cell differentiation models

  • Chick embryo electroporation for in vivo developmental studies

  • CRISPR/Cas9 genome editing in zebrafish for rapid phenotyping

• Neurological Function/Glioma:

  • Patient-derived glioma cell lines

  • Intracranial xenograft models

  • Neurosphere culture systems

  • Brain slice cultures with manipulated HOXC10 expression

Each model system offers distinct advantages for investigating specific aspects of HOXC10 biology. For instance, adipocyte-specific knockout mice have revealed HOXC10's role in suppressing thermogenesis and maintaining white adipocyte identity , while orthotopic colorectal cancer models have demonstrated HOXC10's promotion of metastasis .

How can single-cell techniques be optimized for HOXC10 antibody applications?

Single-cell analysis of HOXC10 requires specific optimization strategies:

• Single-Cell Protein Analysis:

  • CyTOF/mass cytometry using metal-conjugated HOXC10 antibodies

  • Imaging mass cytometry for spatial context within tissues

  • Single-cell Western blotting for protein quantification

  • Optimization of fixation and permeabilization for nuclear antigen preservation

• Spatial Transcriptomics Integration:

  • Combined immunofluorescence for HOXC10 with in situ RNA sequencing

  • Correlation of HOXC10 protein levels with target gene expression

  • Cell type-specific analysis of HOXC10 function

• Technical Considerations:

  • Signal amplification strategies (TSA, branched DNA) for low abundance detection

  • Background reduction through optimized blocking and antibody dilution

  • Computational analysis pipelines for correlating HOXC10 levels with cellular phenotypes

• Validation Approaches:

  • Comparison of antibody-based detection with CRISPR knock-in fluorescent tags

  • Correlation with single-cell RNA-seq data for HOXC10 mRNA

  • Spike-in controls for quantification standardization

These approaches could reveal heterogeneity in HOXC10 expression within tissues that might be missed by bulk analysis methods, particularly in complex environments like tumors and developing tissues where HOXC10 has demonstrated important functional roles .

What are the considerations for developing therapeutic strategies targeting HOXC10?

Development of HOXC10-targeted therapeutics requires multifaceted approaches:

• Target Validation Strategies:

  • Genetic validation through conditional knockout models

  • Temporal control of HOXC10 inhibition using inducible systems

  • Identification of synthetic lethal interactions

  • Patient stratification based on HOXC10 expression/activity

• Therapeutic Approaches:

  • Small molecule inhibitors of HOXC10-DNA binding

  • Disruption of HOXC10 protein-protein interactions

  • Targeted protein degradation approaches (PROTACs)

  • Indirect targeting through CDK7 inhibition (shown to reverse HOXC10-mediated drug resistance)

• Combination Strategies:

  • HOXC10 inhibition with conventional chemotherapy

  • Combining HOXC10 targeting with immunotherapy (given its role in immunosuppressive gene expression)

  • Metabolic interventions in adipose tissue disorders

• Biomarker Development:

  • HOXC10 antibody-based tissue diagnostics for patient stratification

  • Development of activity-based assays for HOXC10 function

  • Identification of downstream markers of HOXC10 inhibition

Research has shown that inhibitors of CDK7 can reverse HOXC10-mediated drug resistance in breast cancer cells by disrupting HOXC10's ability to enhance DNA damage repair . Similarly, targeting HOXC10 in glioma models shows promise for reducing immunosuppressive gene expression and potentially enhancing immunotherapy efficacy .