HOXC11 Antibody

What is HOXC11 and why is it significant in research?

HOXC11 is a sequence-specific transcription factor belonging to the Abd-B homeobox family that functions as part of a developmental regulatory system providing cells with specific positional identities on the anterior-posterior axis. It binds to promoter elements of genes such as lactase-phlorizin hydrolase . Recent research has revealed HOXC11's critical roles in cancer biology, particularly in chemoresistance mechanisms in colorectal cancer and progression of lung adenocarcinoma . The protein's developmental function combined with its emerging role in cancer pathways makes it an important research target for both developmental biology and oncology investigations.

What applications are HOXC11 antibodies commonly used for in research?

HOXC11 antibodies are employed in multiple experimental applications including:

• Western blotting: For detection and quantification of HOXC11 protein expression levels in cell and tissue lysates, typically used at dilutions of 1:1000

• Immunohistochemistry: For visualization of HOXC11 distribution in tissue sections, used at dilutions ranging from 1:50-1:200

• Immunofluorescence: For subcellular localization studies, particularly when investigating HOXC11's presence in different cellular compartments like nuclei and mitochondria, used at concentrations of 0.25-2 μg/mL

• Chromatin immunoprecipitation (ChIP): For analyzing HOXC11 binding to DNA regulatory regions

Each application requires specific optimization of antibody concentration, incubation conditions, and detection methods to ensure specific and sensitive detection of HOXC11.

How can researchers validate HOXC11 antibody specificity?

Validating antibody specificity is critical for reliable experimental outcomes. For HOXC11 antibodies, the following methodological approaches are recommended:

• CRISPR/Cas9 knockout controls: Generate HOXC11 knockout cell lines using CRISPR/Cas9 system as negative controls. Guide RNAs targeting exon 1 of HOXC11 (e.g., gRNA#1: CTACTCCTCCTGCTATGCGG; gRNA#2: GCGCCCCTCTCCTTGCGCGA) can be cloned into vectors like pSpCas9(BB)-2A-Puro (PX459) V2.0 for transfection into target cells .

• Western blot analysis: Compare signals between wildtype and knockout cells using the HOXC11 antibody. A specific antibody will show the expected band (approximately 33.7 kDa) in wildtype cells and absence of this band in knockout cells .

• Immunofluorescence with signal peptide constructs: Use cells transfected with different signal peptide-fused HOXC11 constructs (e.g., NLS-HOXC11, NES-HOXC11, MTS-HOXC11) to confirm antibody detection of HOXC11 in the expected subcellular compartments .

• Peptide competition assay: Pre-incubate the antibody with the immunogen peptide (e.g., STVSSFLPQAPSRQISYPYSAQVPPVREVSYGLEPSGKWHHRNSYSSCYAAADELMHRECLPPSTVTEILMKNEGSYGGHHHPSAPHATPAGFYSSVNKNSVLP for certain antibodies) before application to samples, which should eliminate specific staining .

What are the optimal protocols for studying HOXC11 subcellular localization in cancer cells?

Recent research has identified functional subsets of HOXC11 in different cellular compartments, particularly in the mitochondria of chemoresistant colorectal cancer cells . The following methodological approach is recommended:

• Co-localization studies with organelle markers:

  • Seed cells onto glass coverslips and allow adherence overnight

  • Label mitochondria by incubating cells with 50 nM MitoTracker™ Red CMXRos in culture medium containing reduced FBS (1%) at 37°C for 30 minutes

  • Wash cells with PBS, fix with 4% formaldehyde, and permeabilize with 0.1% Triton X-100

  • Block with 5% FBS in PBST for 1 hour at room temperature

  • Incubate with anti-HOXC11 primary antibody (e.g., Thermo Fisher Scientific, catalog #: TA502570, dilution: 1:1000) overnight at 4°C

  • Incubate with fluorescently labeled secondary antibodies for 1 hour at room temperature

  • Counterstain nuclei with DAPI for 20 minutes at room temperature

  • Image using confocal microscopy with appropriate filter sets

• Subcellular fractionation and Western blotting:

  • Isolate mitochondrial, nuclear, and cytoplasmic fractions using commercial kits

  • Perform Western blotting on each fraction using anti-HOXC11 antibody

  • Include fraction-specific markers (e.g., VDAC for mitochondria, Lamin B for nuclei) to confirm fractionation quality

  • Compare HOXC11 distribution between normal and chemoresistant cancer cells

How can researchers investigate HOXC11's role in regulating mitochondrial function and chemoresistance?

HOXC11 has been shown to regulate mitochondrial function through modulation of mtDNA transcription, impacting chemoresistance in colorectal cancer . A comprehensive experimental approach includes:

• HOXC11 manipulation in cell models:

  • Generate HOXC11 knockout cells using CRISPR/Cas9 system

  • Create stable overexpression models using signal peptide-fused constructs (NLS-HOXC11, NES-HOXC11, MTS-HOXC11) to target HOXC11 to specific compartments

  • Develop chemoresistant cell models through gradual exposure to increasing concentrations of chemotherapeutic agents

• Mitochondrial function assessment:

  • Perform Seahorse mito stress test to evaluate mitochondrial respiratory function

  • Measure mtDNA copy number using real-time PCR

  • Analyze mtDNA transcription levels using RT-PCR for mitochondrial genes

  • Assess mitochondrial membrane potential using fluorescent dyes

• Chemoresistance evaluation:

  • Conduct Sulforhodamine B assay to assess cell viability following treatment with chemotherapeutic drugs

  • Compare IC50 values between wildtype, HOXC11-knockout, and HOXC11-overexpressing cells

  • Perform apoptosis assays to determine if HOXC11 affects drug-induced apoptotic pathways

What is the optimal protocol for HOXC11 chromatin immunoprecipitation to identify its binding sites?

For investigating HOXC11 binding to genomic or mitochondrial DNA, the following ChIP protocol is recommended:

• Mitochondrial ChIP (if studying mitochondrial targets):

  • Isolate mitochondria from cells using a commercial mitochondrial isolation kit

  • Crosslink protein-DNA complexes with 1% formaldehyde at room temperature for 10 minutes

  • Extract and shear mtDNA into smaller fragments via sonication

  • Incubate fragmented mtDNA overnight at 4°C with anti-HOXC11 antibody

  • Capture antibody-bound complexes using protein A/G-coated magnetic beads

  • Wash thoroughly to remove non-specifically bound DNA

  • Elute and purify DNA for subsequent analysis

  • Perform quantitative real-time PCR using primers targeting regions of interest (e.g., mtDNA D-loop region: forward: CACCCCTCACCCACTAGGATAC; reverse: TCCATGGGGACGAGAAGGGATT)

• Nuclear ChIP (if studying genomic targets):

  • Similar protocol as above but starting with whole cells

  • Include appropriate controls: IgG negative control, input samples, and positive control antibodies (e.g., anti-TFAM for mitochondrial targets)

  • Analyze enrichment by qPCR or sequencing (ChIP-seq)

How does HOXC11 expression correlate with cancer prognosis and what methodologies are used to establish this correlation?

HOXC11 expression has been associated with cancer prognosis in multiple tumor types. For investigating this correlation:

What experimental approaches can be used to study HOXC11's transcriptional targets?

As a transcription factor, HOXC11 regulates the expression of various genes. To identify and validate these targets:

• Genome-wide approaches:

  • ChIP-seq: Perform chromatin immunoprecipitation followed by high-throughput sequencing

  • RNA-seq: Compare transcriptomes between HOXC11 wildtype, knockout, and overexpression models

  • ATAC-seq: Analyze chromatin accessibility in relation to HOXC11 binding

• Target validation approaches:

  • Luciferase reporter assays with wild-type and mutated promoter constructs

  • Electrophoretic mobility shift assays (EMSA) to confirm direct binding

  • RT-qPCR and Western blotting to validate expression changes at mRNA and protein levels

Research has identified specific targets of HOXC11, such as its role in regulating mtDNA transcription in colorectal cancer cells and promoting SPHK1 expression in lung adenocarcinoma by directly binding to its promoter region .

What are the critical factors in selecting an appropriate HOXC11 antibody for specific research applications?

When selecting a HOXC11 antibody, researchers should consider:

• Antibody characteristics:

  • Type: Monoclonal (e.g., clone HOX5J232 from Iwai North America Inc.) versus polyclonal (e.g., HPA038291 from Sigma-Aldrich)

  • Host species: Mouse or rabbit, depending on experimental design and compatibility with other antibodies

  • Immunogen: Target-specific epitope (e.g., STVSSFLPQAPSRQISYPYSAQVPPVREVSYGLEPSGKWHHRNSYSSCYAAADELMHRECLPPSTVTEILMKNEGSYGGHHHPSAPHATPAGFYSSVNKNSVLP)

  • Validated applications: Ensure the antibody has been validated for your specific application (WB, IHC, IF)

• Validation status:

  • Look for antibodies with extensive validation data in applications similar to your planned experiments

  • Check for specificity validation using knockout controls

  • Review published literature using the specific antibody

• Application-specific considerations:

  Application	Key Considerations
  Western Blot	Denaturing vs. non-denaturing conditions, blocking agents, detection systems
  Immunohistochemistry	Fixation method, antigen retrieval, detection system
  Immunofluorescence	Fixative compatibility, co-staining possibilities, autofluorescence
  ChIP	Crosslinking efficiency, chromatin fragmentation, antibody specificity

How can researchers address common technical challenges when working with HOXC11 antibodies?

• High background in immunostaining:

  • Optimize blocking conditions (try 5% FBS in PBST for 1 hour at room temperature)

  • Increase washing steps (use at least three 5-minute washes between antibody incubations)

  • Titrate primary antibody concentration (test ranges from 0.25-2 μg/mL for immunofluorescence)

  • Use appropriate negative controls (IgG control, HOXC11 knockout samples)

• Weak or no signal in Western blotting:

  • Ensure protein extraction method preserves HOXC11 (avoid excessive heat)

  • Optimize transfer conditions for proteins in HOXC11's molecular weight range (~33.7 kDa)

  • Try different membrane types (PVDF vs. nitrocellulose)

  • Increase antibody concentration or incubation time

• Conflicting results between antibodies:

  • Verify antibody epitopes and potential isoform recognition

  • Compare monoclonal vs. polyclonal antibodies

  • Use orthogonal detection methods (mRNA expression, tagged constructs)

How can HOXC11 antibodies be utilized to study the protein's role in therapy resistance mechanisms?

Recent research has highlighted HOXC11's role in chemoresistance, particularly in colorectal cancer . To investigate therapy resistance mechanisms:

• Paired sensitive/resistant cell models:

  • Develop matched sensitive and resistant cell lines through gradual drug exposure

  • Compare HOXC11 expression, localization, and target gene regulation between paired lines

  • Use HOXC11 antibodies for Western blotting, immunofluorescence, and ChIP analyses

• Patient-derived samples analysis:

  • Compare HOXC11 expression in pre-treatment versus post-relapse tumor samples

  • Correlate HOXC11 expression with treatment response and survival outcomes

  • Use tissue microarrays and immunohistochemistry with optimized HOXC11 antibodies

• Mechanistic studies:

  • Investigate HOXC11's interaction with drug efflux pumps or pro-survival pathways

  • Study HOXC11's regulation of mitochondrial function in relation to drug resistance

  • Explore combination therapies targeting HOXC11-regulated pathways

What methodological approaches can be used to study post-translational modifications of HOXC11?

Post-translational modifications can significantly affect HOXC11 function, localization, and stability. To investigate these:

• Identification approaches:

  • Immunoprecipitate HOXC11 using validated antibodies followed by mass spectrometry

  • Use modification-specific antibodies (if available) for Western blotting

  • Combine with phosphatase or deubiquitinase treatments to confirm modifications

• Functional impact assessment:

  • Generate site-specific mutants (e.g., phosphomimetic, phosphodeficient)

  • Compare subcellular localization using immunofluorescence with HOXC11 antibodies

  • Assess impact on transcriptional activity using reporter assays

  • Evaluate protein stability and turnover

• Regulation studies:

  • Investigate the role of IKKα in HOXC11 regulation, as recent research suggests a connection to ubiquitination of HOXC11 in lung cancer

  • Study how modifications affect HOXC11's ability to bind mtDNA and nuclear targets