hoxa11b Antibody

What is HOXA11B and how does it differ from HOXA11?

HOXA11B (homeobox A11b) is a zebrafish homolog of the human HOXA11 gene, which encodes a sequence-specific transcription factor involved in embryonic development and spatial patterning. While HOXA11 is found in mammals including humans, mice, and rats, HOXA11B is specifically found in zebrafish (Danio rerio) . Both proteins belong to the Abd-B homeobox family and function as part of a developmental regulatory system that provides cells with specific positional identities on the anterior-posterior axis . The zebrafish HOXA11B protein is predicted to enable DNA-binding transcription factor activity, RNA polymerase II-specific binding activity, and is involved in embryonic skeletal joint morphogenesis .

HOXA11/HOXA11B predominantly localizes to the nucleus, consistent with its function as a transcription factor . Immunofluorescence studies show nuclear staining patterns in various cell types. In human cell lines like BJ fibroblasts, HOXA11 antibody staining reveals specific localization to the nucleoplasm . This nuclear localization is critical for its function in regulating gene expression during development and in disease states.

What are the optimal conditions for HOXA11 antibody-based Western blotting?

For optimal Western blot detection of HOXA11:

• Sample preparation: Cell or tissue lysates should be prepared in standard lysis buffers containing protease inhibitors to prevent protein degradation .

• Protein loading: 5-10 μg of total protein is typically sufficient for detection in cells expressing endogenous levels of HOXA11 .

• Antibody dilution: Most HOXA11 antibodies perform optimally at dilutions between 1:500-1:1000 .

• Expected molecular weight: While the calculated molecular weight of HOXA11 is approximately 34 kDa, the observed band on Western blots typically appears at 30-37 kDa, depending on the cell type and antibody used .

• Positive controls: Mouse colon, rat uterus, and rat kidney tissues show reliable HOXA11 expression and can serve as positive controls . For cell lines, HeLa cells demonstrate detectable levels of HOXA11 .

• Validation: Including immunizing peptide competition controls can confirm antibody specificity, as demonstrated in Western blots of RAW264.7 cell extracts .

What methods are effective for HOXA11 knockdown in functional studies?

Two primary approaches have proven effective for HOXA11 knockdown:

• RNA interference (siRNA):

  • Successfully employed in various cell types including malignant glioma cell lines (U251, U373, and LN18) .

  • Commercially available sequences targeting HOXA11 (such as #SASI-Hs01-00110410, #SASI-Hs01-00110413, and #SASI-Hs01-00110417) have shown efficacy at standard transfection conditions .

  • Cell viability assays 72 hours post-transfection can effectively measure functional outcomes of HOXA11 knockdown .

• Short hairpin RNA (shRNA):

  • Lentiviral vectors expressing shRNA against HOXA11 (e.g., TRCN0000413738 and TRCN0000417739) at a multiplicity of infection of 1 have been used to generate stable knockdown cell lines .

  • Selection in media containing puromycin (2.5 μg/ml) for approximately 2 weeks is typically required to establish stable lines .

  • Control cells should be transfected with blank lentiviral vectors (e.g., pLKO_025) .

Note that synthetic HOXA11 antisense oligonucleotides have shown limited efficacy in blocking HOXA11 protein expression, unlike similar approaches for HOXA10 .

How does HOXA11 expression influence cancer progression and treatment response?

HOXA11 expression has significant implications for cancer progression and treatment response, particularly in:

• Acute Myeloid Leukemia (AML):

  • Higher HOXA11 expression is associated with improved responses to cytarabine (Ara-C) treatment .

  • HOXA11 expression increases cell apoptosis sensitivity, with functional studies demonstrating that HOXA11 knockdown reduces Ara-C sensitivity in vitro .

  • HOXA11 regulates the expression of apoptosis-related genes, including NF-κB inhibitor α, transcription factor p65, and transformation-related protein p53 .

  • Meta-analyses using datasets like Heuser's AML dataset confirmed that chemotherapy responders have higher expression levels of HOXA11 .

• Glioblastoma (GBM):

  • Underexpression of HOXA11 is associated with poor prognosis in GBM patients .

  • Patients with high HOXA11 expression showed significantly longer survival (31±15.3 months) compared to those with low expression (18±7.3 months, p=0.03) .

  • HOXA11 suppression in GBM cell lines decreases the anticancer effects of radiotherapy and/or temozolomide .

  • Five candidate mediators (TGFBR2, CRIM1, TXNIP, DPYSL2, and CRMP1) have been identified that may confer oncologic effects after HOXA11 suppression .

What role does HOXA11/HOXA11B play in embryonic development?

HOXA11 and its zebrafish homolog HOXA11B play critical roles in embryonic development:

• Vertebrate development:

  • HOXA11B is expressed in several developmental structures, including the central nervous system, chondroblast, mesenchyme, pectoral fin bud, and pectoral fin field in zebrafish .

  • In vertebrates, HOXA11 is part of the HOX gene family that regulates embryonic patterning along the anterior-posterior axis .

• Reproductive system development:

  • HOXA11 is essential for female fertility, governing the cyclic development of the adult endometrium and uterus formation during embryogenesis .

  • HOXA11 expression significantly increases during the mid-luteal phase of the menstrual cycle to facilitate blastocyst implantation .

  • In mice, HOXA11 is required for proper development of the female reproductive tract, and studies using targeted gene disruption have demonstrated its crucial role in fertility .

• Skeletal development:

  • Human mutations in HOXA11 are implicated in radioulnar synostosis, highlighting its role in proper bone formation .

  • In zebrafish, HOXA11B is predicted to be involved in embryonic skeletal joint morphogenesis .

How can polycomb-mediated repression studies enhance our understanding of HOXA11B regulation?

Polycomb group (PcG) proteins are important transcriptional repressors that regulate HOX gene expression, including HOXA11B. Recent research using zebrafish models has provided valuable insights:

• Epigenetic regulation:

  • PcG proteins, particularly through Ezh2 (part of Polycomb Repressive Complex 2), are responsible for catalyzing the H3K27me3 repressive mark that regulates HOX gene expression .

  • Zebrafish embryos lacking maternal and zygotic ezh2 (MZezh2 mutants) provide a unique model for studying Polycomb-mediated repression of developmental genes like HOXA11B .

• Methodological approach:

  • ChIP-seq analysis for PcG proteins (Ezh2, Rnf2) and associated histone marks (H3K27me3, H3K4me3) can reveal the epigenetic landscape controlling HOXA11B expression .

  • Multi-omics approaches combining ChIP-seq, transcriptomics, and proteomics provide comprehensive insights into how PcG proteins maintain spatially restricted expression of HOX genes .

• Developmental timing:

  • Polycomb-mediated repression becomes essential immediately after body plan formation to maintain spatially restricted expression of transcription factors like HOXA11B .

  • This timing differs between vertebrate species, highlighting the importance of comparative studies .

What are the challenges in developing species-specific HOXA11B antibodies for zebrafish research?

Developing and validating zebrafish-specific HOXA11B antibodies presents several challenges:

• Sequence homology considerations:

  • While zebrafish HOXA11B shares high sequence homology with human HOXA11 (approximately 93% in some regions) , the specific epitopes may differ.

  • For optimal specificity, immunogens should target unique regions of zebrafish HOXA11B that do not cross-react with other HOX family members .

• Validation in zebrafish models:

  • Zebrafish-specific positive controls are essential for validating antibody specificity.

  • Tissues showing known HOXA11B expression (pectoral fin bud, central nervous system) should be used as positive controls .

  • Transgenic zebrafish expressing tagged versions of HOXA11B can serve as validation tools for antibody specificity.

• Technical approaches:

  • Western blot validation should include molecular weight verification (~34 kDa predicted) and knockout/knockdown controls .

  • Whole-mount immunostaining protocols specific to zebrafish embryos need optimization for penetration and background reduction.

  • Antibodies raised against recombinant zebrafish HOXA11B protein rather than peptides may provide better specificity and sensitivity.

How can researchers effectively distinguish between HOXA11 paralogs in functional studies?

Distinguishing between HOXA11 paralogs (such as human HOXA11 vs. zebrafish HOXA11B, or HOXA11 vs. other HOX family members) requires careful methodological approaches:

• Antibody selection strategies:

  • Use antibodies raised against unique regions that differ between paralogs .

  • Validate antibody specificity through peptide competition assays and testing in systems with known expression patterns .

  • Consider using multiple antibodies targeting different epitopes to confirm findings.

• Gene expression analysis:

  • Design PCR primers or probes that target unique regions to distinguish between paralogs at the mRNA level.

  • Employ RNA-seq analysis with stringent mapping parameters to differentiate closely related transcripts.

  • Consider single-cell approaches to resolve paralog expression at cellular resolution.

• Functional validation:

  • Use paralog-specific knockdown approaches (siRNA, shRNA, or CRISPR-Cas9) targeting unique regions .

  • Rescue experiments with paralog-specific expression constructs can confirm functional specificity.

  • Cross-species complementation studies can reveal functional conservation or divergence between paralogs.

HOXA11 Antibody Specifications Comparison

Survival Data Related to HOXA11 Expression in Cancer

HOXA11 Knockdown Effects on Treatment Response in GBM Cell Lines