HMBOX1 Antibody

What is HMBOX1 and why is it important in research?

HMBOX1 is a novel transcription factor belonging to the homeobox family of genes. It contains a homeobox domain in the N-terminus and an HNF1-N domain in the C-terminus. HMBOX1 is phylogenetically related to HNF1A and HNF1B but possesses an atypical homeo-domain with a 21-amino acid insertion between the second and third helix . The importance of HMBOX1 in research stems from its diverse functions in multiple biological processes, including its role as a potential transcription repressor and its involvement in natural killer (NK) cell regulation. Studies have shown that HMBOX1 negatively regulates NK cell functions by suppressing the NKG2D/DAP10 signaling pathway, making it relevant for immunology research . Additionally, its differential expression in various cancer tissues suggests potential roles in tumor pathobiology .

What types of HMBOX1 antibodies are available for research?

Several types of HMBOX1 antibodies are available for research applications:

• Polyclonal antibodies targeting different regions of HMBOX1:

  • N-terminal region-specific antibodies

  • Full-length protein (AA 1-420) antibodies

  • Mid-region (AA 142-407) antibodies

  • Specific epitope (e.g., AA 197-223, AA 277-326) antibodies

• Host species variations:

  • Rabbit polyclonal antibodies (most common)

  • Mouse polyclonal antibodies

• Application-specific antibodies validated for:

  • Western Blotting (WB)

  • Immunohistochemistry (IHC/IHC-P)

  • Immunocytochemistry (ICC)

  • Immunofluorescence (IF)

  • Immunoprecipitation (IP)

  • ELISA

What is the expression profile of HMBOX1 in normal and cancer tissues?

HMBOX1 shows tissue-specific expression patterns that differ between normal and cancerous states:

This differential expression pattern suggests that HMBOX1 may play distinct roles in different cancer types, potentially serving as a biomarker or therapeutic target in specific contexts .

What are the best experimental conditions for using HMBOX1 antibodies in Western blotting?

For optimal Western blotting using HMBOX1 antibodies, follow these methodological guidelines:

• Sample preparation:

  • Use fresh cell lysates as positive controls

  • Include both HMBOX1-expressing tissues (e.g., kidney) and known low-expressing samples for comparison

  • Standard protein extraction with RIPA buffer containing protease inhibitors is recommended

• Antibody selection and dilution:

  • Select antibodies based on the region of interest (N-terminal antibodies for detecting full-length HMBOX1 and potential splice variants like HMBOX1b)

  • Start with manufacturer's recommended dilution (typically 1:1000) and optimize if needed

  • For polyclonal antibodies, protein A purified antibodies show good specificity

• Detection conditions:

  • Use 1x PBS buffer with 0.09% sodium azide for antibody preparation

  • Avoid repeated freeze-thaw cycles of antibody solutions

  • Store antibodies at -20°C in small aliquots for long-term storage

• Controls:

  • Include positive controls from tissues known to express HMBOX1 (kidney, pancreas)

  • Consider using HMBOX1-overexpressing cell lines as additional positive controls

How can I validate the specificity of HMBOX1 antibodies for my research?

Validating antibody specificity is crucial for reliable HMBOX1 research. Consider these methodological approaches:

• Genetic validation:

  • Compare staining in wild-type cells versus HMBOX1 knockdown cells using siRNA or shRNA

  • Overexpress HMBOX1 in low-expressing cell lines and confirm increased signal

  • Studies have shown that knocking down HMBOX1 expression with lentivirus-delivered shRNA can effectively reduce HMBOX1 levels for validation purposes

• Cross-validation with multiple antibodies:

  • Use antibodies targeting different epitopes (N-term vs. mid-region)

  • Compare results from at least two independent antibodies (e.g., 2A5F4 and 4A4F2 monoclonal antibodies which have been specifically generated against human HMBOX1)

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide

  • Signal should be reduced or eliminated in the presence of the specific peptide

• Molecular weight confirmation:

  • Confirm HMBOX1 detection at the expected molecular weight (~420 amino acids for full-length protein)

  • Check for detection of known splice variants (e.g., HMBOX1b, a 304-amino acid variant without the homeo-domain and C-terminal region)

How should HMBOX1 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling are essential for maintaining HMBOX1 antibody activity:

• Storage recommendations:

  • Store at -20°C for long-term storage

  • For short-term use (up to 1 week), store at 2-8°C

  • Aliquot antibodies into small volumes to prevent repeated freeze-thaw cycles

• Buffer conditions:

  • Most HMBOX1 antibodies are supplied in 1x PBS buffer with 0.09% (w/v) sodium azide and 2% sucrose

  • Maintain these conditions when diluting antibodies

• Safety precautions:

  • Handle with care as antibody solutions contain sodium azide, which is poisonous

  • Only trained staff should handle these reagents

• Reconstitution (if lyophilized):

  • Follow manufacturer's guidelines for reconstitution volumes

  • Allow antibody to fully dissolve before use (gentle inversion, no vortexing)

• Quality control:

  • Monitor antibody performance regularly with positive controls

  • Document lot-to-lot variation by maintaining control samples

How does HMBOX1 expression vary across different cell lines and how might this affect antibody detection?

HMBOX1 expression shows significant variation across different cell types, which can impact experimental design and antibody detection:

• Expression patterns:

  • Primary human NK cells: High expression of HMBOX1

  • NK cell lines (NK-92, NKL): Much lower expression than primary NK cells

  • Human PBMCs: Lower expression than primary NK cells

  • Expression levels appear to correlate with cell activation state

• Factors affecting expression:

  • Cell activation status: Resting primary NK cells show high HMBOX1 expression while activated NK cells show reduced expression

  • IL-2 stimulation: Treatment with IL-2 (100 U) for 6 hours reduces HMBOX1 expression in primary NK cells

  • IL-2 starvation: Removal of IL-2 from NK-92 cells for 24 hours slightly increases HMBOX1 expression

• Impact on antibody detection:

  • Higher antibody concentrations may be needed for cells with low HMBOX1 expression

  • Longer exposure times might be required for Western blot detection in low-expressing samples

  • Consider enrichment strategies (e.g., immunoprecipitation) for cell lines with limited expression

• Experimental considerations:

  • Include appropriate positive controls (e.g., primary NK cells for high expression)

  • Consider the activation state of cells when interpreting results

  • Factor in the possibility of detecting splice variants (e.g., HMBOX1b) in different cell types

What functional assays can be used alongside HMBOX1 antibodies to study its role in NK cell regulation?

To investigate HMBOX1's role in NK cell regulation, combine antibody-based detection with these functional assays:

• Cytotoxicity assays:

  • Standard chromium release assay against target cells (e.g., K562)

  • Flow cytometry-based cytotoxicity assays

  • Research has shown that overexpression of HMBOX1 inhibits NK cell cytotoxicity by approximately 15-30%

• Degranulation assays:

  • CD107a (LAMP-1) surface expression by flow cytometry

  • HMBOX1 overexpression significantly reduces CD107a levels in NK cells in response to target cell stimulation

• Cytolytic protein expression:

  • Measure perforin and granzyme production by Western blot or intracellular flow cytometry

  • HMBOX1 overexpression decreases expression levels of these cytolytic proteins

• Receptor expression analysis:

  • Evaluate NKG2D surface expression by flow cytometry

  • Assess DAP10 expression levels by Western blot

  • HMBOX1 negatively regulates both NKG2D expression and NKG2D/DAP10 signaling pathway activation

• Signaling pathway analysis:

  • Western blot analysis of phosphorylated signaling proteins downstream of NKG2D/DAP10

  • Immunoprecipitation to detect protein-protein interactions in the signaling cascade

How can HMBOX1 antibodies be used to study its differential expression in cancer tissues?

HMBOX1 antibodies can be valuable tools for investigating differential expression in cancer tissues:

• Immunohistochemistry (IHC) applications:

  • Use validated antibodies for IHC or IHC-P (paraffin-embedded tissues)

  • Compare expression between tumor tissue and adjacent normal tissue

  • Evaluate cellular localization (nuclear vs. cytoplasmic)

• Tissue microarray (TMA) analysis:

  • Examine HMBOX1 expression across multiple tumor samples simultaneously

  • Correlate expression with clinicopathological features

  • Studies have used specific monoclonal antibodies (2A5F4 and 4A4F2) successfully for such analyses

• Quantitative analysis:

  • Use digital image analysis for quantifying staining intensity

  • Consider H-score or other semi-quantitative scoring systems

  • Stratify samples based on expression levels for correlation with clinical outcomes

• Tissue-specific considerations:

  • Kidney: Focus on renal tubule expression in normal tissue versus clear-cell carcinoma

  • Liver: Compare the dramatically decreased expression in liver cancer versus normal tissue

  • Pancreas: Evaluate the high expression in both cancer and adjacent normal tissue

What experimental approaches can resolve contradictory findings when using different HMBOX1 antibodies?

When facing contradictory results with different HMBOX1 antibodies, implement these methodological strategies:

• Epitope mapping and antibody characterization:

  • Determine the exact binding regions of each antibody

  • Consider potential epitope masking due to protein-protein interactions

  • Use antibodies targeting different regions (N-term, mid-region, C-term) of HMBOX1

• Validation with orthogonal methods:

  • Complement antibody-based detection with mRNA analysis (qRT-PCR)

  • Use mass spectrometry for protein identification and quantification

  • Employ CRISPR-Cas9 gene editing to create true negative controls

• Analysis of splice variants:

  • Design experiments to distinguish between full-length HMBOX1 and splice variants like HMBOX1b

  • Use antibodies that can specifically detect one isoform but not others

  • Compare results with isoform-specific PCR primers

• Control for post-translational modifications:

  • Consider whether phosphorylation or other modifications might affect antibody binding

  • Use phosphatase treatment to eliminate phosphorylation-dependent epitopes

  • Investigate potential protein degradation with protease inhibitors

• Systematic documentation of conditions:

  • Record detailed experimental conditions for each antibody

  • Standardize protocols when comparing multiple antibodies

  • Create a decision tree for selecting the most appropriate antibody based on the application

How can researchers leverage HMBOX1 antibodies to elucidate its transcriptional repressor functions?

To investigate HMBOX1's role as a transcriptional repressor, combine antibody techniques with these approaches:

• Chromatin immunoprecipitation (ChIP) assays:

  • Use HMBOX1 antibodies to identify DNA binding sites genome-wide

  • Validate high-affinity binding with focused ChIP-qPCR

  • Previous studies suggest HMBOX1 may be a ubiquitous transcription repressor expressed in most human tissues

• Co-immunoprecipitation (Co-IP) studies:

  • Identify protein interaction partners that contribute to transcriptional repression

  • Focus on potential interactions with known transcriptional co-repressors

  • Use antibodies validated for immunoprecipitation applications

• Reporter gene assays:

  • Use luciferase or other reporter systems to measure transcriptional repression

  • Compare wild-type HMBOX1 with mutant variants (e.g., HMBOX1b retains only faint transcriptional repressive activity)

  • Design experiments similar to the pM-HMBOX1 and pGAL4 5tkLUC co-transfection system previously described

• Subcellular localization studies:

  • Determine nuclear versus cytoplasmic distribution using fractionation and immunoblotting

  • Employ immunofluorescence to visualize localization patterns

  • HMBOX1 has been detected in both cytoplasm and nucleus of various tissues

• Gene expression analysis after modulation:

  • Identify genes regulated by HMBOX1 using RNA-seq after overexpression or knockdown

  • Validate direct targets by combining with ChIP data

  • Focus on genes involved in NK cell function as potential targets

What are the key considerations when designing experiments to study HMBOX1's role in different cancer types?

When investigating HMBOX1 in cancer contexts, consider these research design elements:

• Tissue-specific expression patterns:

  • Design studies acknowledging the differential expression across cancer types:

    • Increased expression in kidney clear-cell carcinoma

    • Similar expression in pancreatic cancer and normal tissue

    • Decreased expression in liver cancer compared to normal tissue

• Cell line selection:

  • Choose cell lines representing different cancer types with known HMBOX1 expression patterns

  • Include both high-expressing and low-expressing lines for comparison

  • Validate expression levels in cell lines before conducting functional studies

• Functional assessments:

  • Proliferation assays after HMBOX1 modulation (overexpression/knockdown)

  • Migration/invasion assays to assess metastatic potential

  • Apoptosis assays to determine cell survival effects

  • Consider the impact of HMBOX1 on immune surveillance (particularly NK cell interactions)

• In vivo models:

  • Develop xenograft models with HMBOX1-modulated cancer cells

  • Consider tissue-specific transgenic or knockout models

  • Evaluate both tumor growth and immune infiltration

• Clinical correlation:

  • Design tissue microarray studies with sufficient statistical power

  • Correlate HMBOX1 expression with patient outcomes and clinicopathological features

  • Consider potential therapeutic implications based on tissue-specific expression patterns

What emerging technologies might enhance the utility of HMBOX1 antibodies in research?

Several cutting-edge technologies could expand the applications of HMBOX1 antibodies:

• Single-cell antibody-based techniques:

  • Single-cell Western blotting for heterogeneity analysis

  • Mass cytometry (CyTOF) incorporating HMBOX1 antibodies

  • Imaging mass cytometry for spatial context in tissues

• Proximity labeling approaches:

  • BioID or APEX2 fusions with HMBOX1 to identify proximal interactors

  • Proximity ligation assays to detect protein-protein interactions in situ

  • Combine with antibody-based detection for validation

• Live-cell imaging applications:

  • Development of cell-permeable antibody fragments (nanobodies)

  • Fluorescent labeling strategies for real-time tracking

  • Correlative light and electron microscopy for ultrastructural localization

• Therapeutic development:

  • Antibody-drug conjugates targeting surface-expressed HMBOX1 in relevant cancers

  • Intrabodies to modulate HMBOX1 function in specific cellular compartments

  • CAR-T approaches for cancers with high HMBOX1 expression

• Spatial transcriptomics integration:

  • Combine antibody-based protein detection with spatial transcriptomics

  • Correlate protein expression with transcriptional landscapes at tissue level

  • Develop multiplex approaches for simultaneous detection of HMBOX1 and its targets

How might understanding HMBOX1's role in NK cells inform potential immunotherapeutic approaches?

HMBOX1's function as a negative regulator of NK cells suggests several immunotherapeutic strategies:

• NK cell potentiation strategies:

  • Targeted inhibition of HMBOX1 to enhance NK cell activity

  • Development of small molecule inhibitors or biologics that disrupt HMBOX1 function

  • HMBOX1 knockdown has been shown to enhance NK cell cytolytic function

• NKG2D/DAP10 pathway modulation:

  • Approaches to counteract HMBOX1's negative regulation of NKG2D expression

  • Enhancing DAP10 signaling to overcome HMBOX1-mediated suppression

  • Combination with existing NKG2D-based immunotherapies

• Adoptive NK cell therapy optimization:

  • Ex vivo modification of NK cells to reduce HMBOX1 expression

  • Selection of NK cells with naturally lower HMBOX1 levels

  • Manipulation of IL-2 signaling, which reduces HMBOX1 expression

• Predictive biomarkers:

  • Evaluation of HMBOX1 expression as a predictor of response to immunotherapy

  • Stratification of patients based on tumor and immune cell HMBOX1 expression

  • Development of companion diagnostics for HMBOX1-targeted therapies

• Combination therapy approaches:

  • Rational design of combinations targeting HMBOX1 alongside immune checkpoint inhibitors

  • Sequential therapy strategies based on HMBOX1 expression dynamics

  • Tumor-specific approaches considering differential expression across cancer types