HOXB2 Antibody

What is HOXB2 and what are its primary functions in cellular biology?

HOXB2 is a sequence-specific transcription factor belonging to the homeobox protein family. It functions as part of a developmental regulatory system that provides cells with specific positional identities on the anterior-posterior axis . As a homeoprotein, HOXB2 acts as a master regulator of embryonic development . Recent research demonstrates that HOXB2 plays pivotal roles in multiple cancer types, including breast, lung, cervical, and esophageal cancers, where it can function as either an oncogene or tumor suppressor depending on the cancer context . At the molecular level, HOXB2 exerts its regulatory effects by binding to specific DNA sequences in the promoter regions of target genes such as MATN3 and ECM2, thereby controlling their transcription .

What types of HOXB2 antibodies are currently available for research applications?

Based on the available research data, rabbit polyclonal antibodies against HOXB2 represent the primary type utilized in scientific investigations. One well-characterized example is ab220390 from Abcam, a rabbit polyclonal antibody that has been validated for Western blot (WB) and immunocytochemistry/immunofluorescence (ICC/IF) applications with human samples . This particular antibody targets a recombinant fragment protein within the human HOXB2 sequence, specifically amino acids 50-100 . Other HOXB2 antibodies suitable for chromatin immunoprecipitation (ChIP) assays and immunohistochemistry have also been documented in research studies examining HOXB2's role in cancer progression .

What is the expected molecular weight of HOXB2 protein in Western blot analyses?

According to technical data from validated anti-HOXB2 antibodies such as ab220390, the predicted molecular weight for HOXB2 protein in Western blot applications is approximately 37 kDa . This has been confirmed through experimental validation using human cell lines including HEK-293 (Human epithelial cell line from embryonic kidney) and PC3 (Human prostate adenocarcinoma cell line) . When performing Western blot analysis, researchers should expect to observe distinct bands at this molecular weight position when using properly validated anti-HOXB2 antibodies.

How is HOXB2 implicated in the progression of triple-negative breast cancer?

HOXB2 has been identified as a significant suppressor of triple-negative breast cancer (TNBC) progression. Unlike other breast cancer subtypes, HOXB2 is specifically downregulated in the aggressive TNBC molecular subtype . Research has demonstrated that reduced expression of HOXB2 correlates strongly with enhanced metastatic capabilities of TNBC cells, particularly through promotion of epithelial-to-mesenchymal transition (EMT) . Mechanistically, HOXB2 restrains TNBC aggressiveness by regulating extracellular matrix (ECM) organization. Chromatin immunoprecipitation studies have revealed that HOXB2 directly binds to the promoter regions of ECM-related genes including MATN3 and ECM2, thereby regulating their transcription levels . Importantly, forced expression of HOXB2 in experimental models effectively prevented TNBC progression and metastasis in mouse xenograft models, highlighting its potential therapeutic significance .

What prognostic value does HOXB2 expression offer in glioma research?

HOXB2 has been conclusively identified as an independent prognostic biomarker in glioma patients through comprehensive analysis of large clinical datasets. Expression analysis demonstrates that HOXB2 levels positively correlate with tumor grade, with higher expression observed in high-grade gliomas . Additionally, HOXB2 expression is significantly enriched in patients with isocitrate dehydrogenase 1 (IDH1) wild-type tumors and in patients older than 41 years . Survival analyses using Kaplan-Meier methods have consistently shown that patients with high HOXB2 expression experience significantly worse prognosis in both the Chinese Glioma Genome Atlas (CGGA) and The Cancer Genome Atlas (TCGA) datasets . Multivariate Cox regression analyses further confirm high HOXB2 expression as an independent risk factor for poor prognosis (HR = 1.237; 95% CI: 1.094–1.398; P < 0.001) as shown in Table 1 .

Table 1: Multivariate Analysis of HOXB2 as a Prognostic Factor in Glioma

What methodologies exist for manipulating HOXB2 expression in experimental cancer models?

Multiple validated approaches exist for modulating HOXB2 expression levels in experimental cancer models. For overexpression studies, cells can be transfected with GFP-HOXB2 plasmid (utilizing vectors such as pCMV6-AC-GFP backbone from Origene) using established transfection reagents like Attractene Transfection Reagent (Qiagen) . To generate stable cell lines constitutively overexpressing HOXB2, transfected cells should be treated with selection antibiotics such as neomycin (0.5 mg/ml) for approximately 2 weeks . For transient knockdown experiments, HOXB2-specific siRNAs can be transfected at an optimal concentration of 40 nM for 48 hours to effectively reduce endogenous HOXB2 expression . These complementary approaches enable researchers to investigate both gain-of-function and loss-of-function phenotypes associated with HOXB2 in cancer progression.

Which experimental applications have been validated for HOXB2 antibodies?

HOXB2 antibodies have been validated for multiple experimental applications critical for cancer research and molecular biology investigations. The anti-HOXB2 antibody ab220390 has been specifically validated for Western blot (WB) analyses at concentrations as low as 0.04 μg/ml and immunocytochemistry/immunofluorescence (ICC/IF) applications at 4 μg/ml concentrations with human samples . Beyond these applications, HOXB2 antibodies have been successfully employed in chromatin immunoprecipitation (ChIP) assays to investigate the binding of HOXB2 to promoter regions of target genes such as MATN3 and ECM2 . Additionally, HOXB2 antibodies have proven effective in immunohistochemistry (IHC) analyses of tissue samples, enabling evaluation of HOXB2 expression in patient-derived materials . These diverse applications collectively enable comprehensive investigation of HOXB2's expression patterns and molecular functions.

What is the optimal protocol for Western blot analysis using HOXB2 antibodies?

For optimal Western blot detection of HOXB2 protein, the following methodological approach is recommended: Prepare cell lysates from appropriate human cell lines (such as HEK-293 or PC3) through standard lysis protocols . After determining protein concentration, load equal amounts of cell lysate onto 8% SDS-PAGE gels and perform electrophoresis and transfer according to standard protocols . For primary antibody incubation, use anti-HOXB2 antibody (such as ab220390) at a concentration of 0.04 μg/ml . For loading controls, antibodies against β-Actin (1:10000, ab6276, Abcam) or GAPDH (1:10000, ab181602, Abcam) are recommended . Following appropriate secondary antibody incubation and washing steps, proceed with chemiluminescent detection. The expected molecular weight for HOXB2 is approximately 37 kDa . Optimization of antibody concentration may be necessary depending on the specific cell type and expression level of HOXB2.

How should chromatin immunoprecipitation (ChIP) assays be performed with HOXB2 antibodies?

Chromatin immunoprecipitation using HOXB2 antibodies requires careful methodological planning. The protocol should begin with standard chromatin preparation through crosslinking, sonication, and pre-clearing steps . For the immunoprecipitation step, use anti-HOXB2 antibody (such as ab220390, Abcam) . Control immunoprecipitations should be performed using histone modification-specific antibodies (anti-H3K4me3, ab1012; anti-H3K27me3, ab6002; anti-H3K9ac, ab12179) and non-immune mouse IgG (sc2025, Santa Cruz) as negative control . Following immunoprecipitation and washing steps, reverse crosslinking and DNA purification should be performed according to standard protocols. The isolated DNA can then be analyzed by real-time qPCR using primers specific to the promoter regions of suspected HOXB2 target genes . ChIP-PCR data should be presented as percentage of input following normalization with IgG to accurately quantify enrichment of HOXB2 binding at specific genomic regions .

What are the critical considerations for immunohistochemical detection of HOXB2 in tissue samples?

Immunohistochemical detection of HOXB2 in tissue samples requires attention to several critical methodological aspects. First, appropriate antigen retrieval methods must be employed based on tissue fixation procedures. For HOXB2 detection, anti-HOXB2 antibody should be used at an optimal dilution of 1:200 in phosphate-buffered saline with Tween 20 (PBST) buffer . Following primary antibody incubation, DAB (3,3'-diaminobenzidine) staining should be carefully monitored and carried out until the tissue sections develop appropriate brown color indicating HOXB2 expression . Tissue samples should then undergo sequential dehydration with increasing ethanol concentrations (70%, 85%, 95%, and 100%) followed by clearing with xylene . For quantitative analysis, DAB signals can be scored using image analysis software such as ImageJ, with expression levels classified based on signal intensity and area measurements . Appropriate negative controls (omitting primary antibody) and positive controls (tissues known to express HOXB2) should be included to validate staining specificity.

How can researchers identify and validate transcriptional targets of HOXB2?

Identifying and validating transcriptional targets of HOXB2 requires a multi-faceted experimental approach. Initially, potential targets can be identified through chromatin immunoprecipitation followed by sequencing (ChIP-seq) using anti-HOXB2 antibodies to map genome-wide binding sites . Candidate targets should then be validated through targeted ChIP-qPCR assays to confirm HOXB2 binding to specific promoter regions. To establish direct transcriptional regulation, dual luciferase reporter assays should be employed by cloning genomic DNA fragments containing the promoter regions of candidate target genes (such as MATN3 and ECM2) into reporter vectors like pGL3-Basic . These constructs should be co-transfected with HOXB2 expression vectors into appropriate cell lines (such as HEK293T), alongside Renilla luciferase vector as internal control . Changes in target gene expression upon HOXB2 modulation should be confirmed at both mRNA level (using qRT-PCR) and protein level (using Western blot) . Finally, functional validation through rescue experiments can establish the biological relevance of HOXB2-target gene regulatory relationships.

What methodological approaches should be used to investigate HOXB2's role in epithelial-to-mesenchymal transition?

Investigation of HOXB2's role in epithelial-to-mesenchymal transition (EMT) requires comprehensive experimental strategies addressing both molecular mechanisms and functional outcomes. First, establish appropriate cellular models by modulating HOXB2 expression (overexpression or knockdown) in relevant epithelial cancer cell lines . Assess changes in EMT marker expression through Western blot analysis using antibodies against epithelial markers such as E-cadherin (1:10000, ab40772, Abcam) and integrin β4 (1:2000, ab133682, Abcam), along with mesenchymal markers including N-cadherin (1:1000, ab18203, Abcam) and vimentin (1:5000, ab92547, Abcam) .

To evaluate functional consequences, perform migration assays (wound healing/scratch assays) and invasion assays (transwell chambers coated with Matrigel) . Morphological changes should be documented through phase-contrast microscopy. For mechanistic insights, investigate alterations in EMT-related signaling pathways (TGF-β, Wnt/β-catenin) upon HOXB2 modulation. Additionally, examine changes in EMT-inducing transcription factors (Snail, Slug, ZEB1/2) through qRT-PCR and Western blot analyses. Finally, validate findings through in vivo metastasis models to confirm the physiological relevance of HOXB2-mediated regulation of EMT.

How should researchers integrate computational and experimental approaches to study HOXB2 in cancer?

Integration of computational and experimental approaches provides a powerful framework for comprehensive investigation of HOXB2 in cancer. Begin with in silico analyses of large-scale patient datasets (such as TCGA, CGGA, METABRIC) to identify cancer types and molecular subtypes with significant HOXB2 expression alterations . Perform differential expression analysis to compare HOXB2 levels across normal tissues, primary tumors, and metastatic samples. Use survival analysis methods (Kaplan-Meier, Cox regression) to establish prognostic significance of HOXB2 expression .

Employ gene set enrichment analysis (GSEA) and pathway analysis to identify biological processes and signaling pathways associated with HOXB2 expression patterns. Use motif analysis to predict potential HOXB2 binding sites across the genome. Following computational predictions, validate key findings through targeted experimental approaches including modulation of HOXB2 expression in appropriate cell line models, followed by functional assays (proliferation, migration, invasion) . For mechanistic insights, perform ChIP-seq to map genome-wide HOXB2 binding sites and RNA-seq to identify transcriptional changes upon HOXB2 modulation. Finally, validate clinical relevance through tissue microarray analyses of patient samples using immunohistochemistry with anti-HOXB2 antibodies.

What considerations are important when interpreting contradictory results regarding HOXB2's role across different cancer types?

When faced with apparently contradictory results regarding HOXB2's role across different cancer types, researchers should consider several critical factors. First, evaluate the tissue context specificity, as HOXB2 functions can vary dramatically between tissue types due to different cellular environments and co-factor availability . For example, HOXB2 acts as a tumor suppressor in triple-negative breast cancer but may have oncogenic functions in other cancer types .

Second, consider the genetic background of experimental models, including mutation status of key cancer drivers (p53, PTEN, RAS) that may interact with HOXB2 signaling. Third, examine methodological differences between studies, including antibody specificity, experimental approaches for manipulation of HOXB2 expression, and endpoint measurements. Fourth, assess the potential role of post-translational modifications that might alter HOXB2 function without affecting expression levels. Fifth, investigate lncRNAs that may regulate HOXB2 function, such as HOXB-AS1 .

Finally, determine whether discrepancies reflect different stages of cancer progression, as HOXB2's role may evolve from early tumorigenesis to metastatic spread. Resolving these contradictions requires careful experimental design with appropriate controls, multiple complementary approaches, and validation across independent patient cohorts.

What are the most common technical challenges when using HOXB2 antibodies and how can they be addressed?

When working with HOXB2 antibodies, researchers frequently encounter several technical challenges that require systematic troubleshooting. For Western blot applications, non-specific bands may appear due to antibody cross-reactivity. This can be addressed by optimizing antibody concentration (starting with 0.04 μg/ml for ab220390) , increasing washing stringency, and using alternative blocking agents (5% BSA versus milk). If signal intensity is low, consider increasing protein load, extending primary antibody incubation time, or using enhanced chemiluminescence detection systems.

For immunohistochemistry applications, high background staining can be minimized by optimizing antibody dilution (1:200 recommended for ab220390) , extending blocking steps, and using more stringent washing. False negative results may occur due to ineffective antigen retrieval; try multiple methods (heat-induced epitope retrieval in citrate buffer pH 6.0 or EDTA buffer pH 9.0) to determine optimal conditions for HOXB2 detection.

In ChIP assays, low enrichment may result from inefficient chromatin shearing or immunoprecipitation. Optimize sonication conditions to achieve 200-500bp fragments and increase antibody amounts for immunoprecipitation. Additionally, ensure that PCR primers are designed to amplify regions containing predicted HOXB2 binding motifs for maximum sensitivity .

How can researchers validate the specificity of HOXB2 antibodies for their experimental system?

Validating HOXB2 antibody specificity is crucial for generating reliable experimental data. Begin with positive and negative control samples: use cell lines known to express HOXB2 (such as HEK-293) as positive controls and either HOXB2-knockout cells or cells treated with HOXB2-siRNA as negative controls . When analyzing Western blots, confirm that the detected band appears at the expected molecular weight of approximately 37 kDa .

Perform peptide competition assays by pre-incubating the antibody with excess immunizing peptide before application to samples; specific signals should be blocked while non-specific signals will remain. For immunocytochemistry/immunofluorescence applications, validate specificity by demonstrating co-localization with GFP signal in cells transfected with GFP-HOXB2 fusion constructs .

Consider orthogonal validation by using multiple antibodies targeting different epitopes of HOXB2 and confirming consistent results. Additionally, correlate protein detection with mRNA expression using RT-qPCR. For ChIP applications, validate antibody specificity by demonstrating enrichment of known HOXB2 target sites (such as MATN3 and ECM2 promoter regions) compared to control regions and confirming loss of enrichment in HOXB2-knockdown conditions .