INHBB Antibody

What is INHBB and what biological functions does it serve in research contexts?

INHBB (Inhibin Beta B) encodes a protein subunit that plays multiple significant roles in physiology. The inhibin beta B subunit joins with the alpha subunit to form inhibin B, which functions as a pituitary FSH secretion inhibitor. Additionally, the beta B subunit can form a homodimer called activin B, or combine with the beta A subunit to create a heterodimer known as activin AB, both of which stimulate FSH secretion .

Research has shown that inhibin regulates gonadal stromal cell proliferation negatively and exhibits tumor-suppressor activity. Importantly, inhibin may function both as a growth/differentiation factor and as a hormone, as its expression in gonadal and various extragonadal tissues can vary significantly in a tissue-specific manner .

What applications are INHBB antibodies validated for in research settings?

Based on current research protocols, INHBB antibodies have been validated for multiple applications:

Many commercially available antibodies show reactivity across human, mouse, and rat samples, making them versatile for comparative studies across species .

What methodological considerations should researchers address when designing experiments with INHBB antibodies?

When designing experiments using INHBB antibodies, researchers should consider:

• Sample preparation: For Western blotting, proper cell lysis and protein extraction protocols are critical. For immunohistochemistry, fixation methods significantly affect epitope availability .

• Controls: Use appropriate positive controls. According to published research, U87, A549, A431, and Hela whole cell lysates have been validated for Western blots .

• Antibody specificity: Consider that some earlier-generation antibodies to the betaB subunit (such as C5) required pre-treatment of samples with hydrogen peroxide to oxidize methionines in the epitope for full immunoreactivity. Newer antibodies like 46A/F have improved specificity without requiring sample pre-treatment .

• Cross-reactivity assessment: Some older assays demonstrated cross-reactivity with inhibin A, although recent findings suggest this cross-reaction is ten-fold less than previously reported .

How does INHBB expression correlate with immune cell infiltration in cancer microenvironments?

Recent research has revealed significant correlations between INHBB expression and immune cell infiltration in cancer microenvironments, particularly in gastric cancer:

• INHBB expression strongly correlates with macrophage infiltration (R = 0.2, p = 2e-05), endothelial cells (R = 0.38, p < 2.2e-16), and cancer-associated fibroblasts (R = 0.25, p < 9.3e-08) in gastric cancer samples .

• High INHBB expression coupled with high macrophage infiltration correlates with worse prognosis in gastric cancer patients (HR = 1.89, p = 0.0144) .

• INHBB expression shows significant correlation with immune cells such as Th2 cells (p < 0.001), NK cells (p < 0.001), and Tem (p < 0.001) .

• INHBB expression is negatively correlated with several chemokines and chemokine receptors, including CXCL3, CXCL10, and CXC11, which play roles in immune cell recruitment .

These findings suggest that INHBB antibodies are valuable tools for studying the complex interactions between cancer cells and the tumor immune microenvironment.

What challenges exist in developing specific antibodies against INHBB?

Developing highly specific antibodies against INHBB presents several significant challenges:

• Conservation across species: The betaB subunit is highly conserved between species, making it difficult to generate strong immune responses when immunizing animals .

• Cross-reactivity: Earlier antibodies showed cross-reactivity with inhibin A, leading to complications in assay specificity. This necessitated careful validation to ensure accurate measurements .

• Epitope accessibility: Some epitopes in the INHBB protein may require special treatment for optimal antibody binding. For example, the C5 antibody required hydrogen peroxide pre-treatment to oxidize methionines in the epitope for full immunoreactivity .

• Varying expression levels: INHBB expression varies significantly across different tissues and disease states, requiring antibodies with broad dynamic ranges for detection .

Newer approaches, such as the RIMMS (Repetitive Immunizations at Multiple Sites) method of immunization with recombinant X. laevis activin B, have shown success in developing highly specific antibodies in shorter time periods .

How can INHBB antibodies be utilized to investigate the role of INHBB in cancer progression?

INHBB antibodies serve as powerful tools for investigating INHBB's role in cancer progression through several methodological approaches:

• Immunohistochemistry for clinical correlation: Using INHBB antibodies for IHC staining of cancer tissue microarrays allows researchers to correlate expression levels with clinical features and patient outcomes. For example, research has shown that INHBB expression correlates with invasion depth (OR: 1.78, 95% CI: 1.16-2.75, p = 0.008), lymph node metastasis (OR: 1.80, 95% CI: 1.27-2.55, p < 0.001), and TNM stage in colorectal cancer .

• Functional studies with knockdown models: Researchers have used shRNA to knock down INHBB expression in cancer cell lines, then evaluated phenotypic changes using functional assays for cellular adhesion, invasiveness, and migration. For example, in oral squamous cell carcinoma, shINHBB cells showed increased adhesion and decreased invasiveness and migration compared to control cells .

• Rescue experiments with recombinant protein: Treatment of INHBB-knockdown cells with recombinant human activin B protein (50 ng/ml) reverted the cellular phenotype, confirming the specificity of INHBB's role in modulating cellular behavior .

• Expression analysis coupled with survival data: INHBB antibodies can be used to stratify patients based on expression levels, which can then be correlated with survival outcomes to establish prognostic value .

What criteria should researchers use when selecting an INHBB antibody for their specific research application?

When selecting an INHBB antibody for specific research applications, researchers should consider:

• Target epitope location: Some antibodies target different regions of the INHBB protein. For example, some polyclonal antibodies are raised against synthetic peptides of human INHBB, while others target recombinant proteins representing specific amino acid sequences (e.g., 295-405AA) .

• Validated applications: Ensure the antibody has been validated for your specific application. For instance, some antibodies work well for Western blot but may not be optimal for immunohistochemistry on paraffin-embedded tissues .

• Species reactivity: Verify the species cross-reactivity. Many commercial INHBB antibodies react with human, mouse, and rat samples, but reactivity should be confirmed for your specific experimental model .

• Clonality consideration:

  • Polyclonal antibodies: Offer broader epitope recognition but potentially more background

  • Monoclonal antibodies: Provide higher specificity but may be more sensitive to epitope modifications

• Purification method: Antibodies purified by affinity methods typically offer higher specificity. For example, Protein G purified antibodies with >95% purity are available for research applications .

What protocol modifications improve detection of INHBB in challenging samples?

For challenging samples with low INHBB expression or high background, consider these methodological modifications:

• For immunohistochemistry:

  • Optimize antigen retrieval: Microwave antigen repair has been successfully used in INHBB detection protocols .

  • Blocking optimization: Incubation with hydrogen peroxide solution at room temperature for 10 minutes effectively blocks endogenous peroxidase activity .

  • Signal amplification: Using DAB (3,3'-diaminobenzidine) for staining followed by hematoxylin counterstaining has been effective for visualizing INHBB expression .

• For Western blotting:

  • Sample preparation: Proper cell lysis and protein extraction protocols are critical for detecting INHBB.

  • Loading controls: Use appropriate loading controls to normalize for protein loading variations.

  • Detection systems: Enhanced chemiluminescence systems may improve sensitivity for low-abundance INHBB detection.

• For immunofluorescence:

  • Secondary antibody selection: Using highly cross-adsorbed secondary antibodies reduces background.

  • Confocal microscopy: Analyzing samples with confocal microscopy and FluoView Software has been effective for INHBB visualization in cellular contexts .

How should researchers quantify INHBB expression in tissue samples?

For accurate quantification of INHBB expression in tissue samples, researchers have employed various methodological approaches:

• For immunohistochemistry quantification:

  • Scoring system: Calculate immunostaining index based on both staining intensity (0-3 scale) and positive staining rate (0-100%) .

  • Threshold determination: Use a defined cutoff value (e.g., >140% has been used to distinguish high vs. low expression) .

  • Digital analysis: Software like IHC Profiler (https://sourceforge.net/projects/ihcprofiler/) can be used for objective quantification .

  • ROC curve analysis: This approach has been used to determine optimal cutoff points for clinical classification .

• For mRNA expression quantification:

  • qRT-PCR protocol: Using primers specific to INHBB (e.g., 5'-ATCAGCTTCGCCGAGACA-3' forward and 5'-GGTTGCCTTCGTTGGAGAT-3' reverse) with GAPDH as reference gene .

  • Median thresholds: Using median expression values to distinguish between high and low expression groups for statistical analyses .

• For correlation with clinical outcomes:

  • Kaplan-Meier analysis: Used to evaluate survival rates between high and low INHBB-expressing groups .

  • Cox regression analysis: Both univariate and multivariate analyses help identify the importance of INHBB as a prognostic factor .

How does INHBB function as a prognostic biomarker in different cancer types?

INHBB has emerged as a significant prognostic biomarker across multiple cancer types, with distinct mechanisms and implications:

These findings highlight the value of INHBB antibodies in cancer prognostication research across multiple tumor types.

What molecular pathways does INHBB influence in the tumor microenvironment?

Research utilizing INHBB antibodies has revealed several key molecular pathways influenced by INHBB in the tumor microenvironment:

• Immune regulation pathways:

  • INHBB expression correlates with macrophage infiltration and affects patient prognosis .

  • INHBB negatively correlates with expression of chemokines CXCL3, CXCL10, and CXCL11, which are involved in immune cell recruitment .

  • INHBB expression has associations with immunoinhibitors (TGFB1, TGFBR1) and immunostimulators (CD276, CXCL12, ENTPD1) .

• Cancer-associated signaling pathways:

  • Gene Set Enrichment Analysis (GSEA) identified INHBB's involvement in epithelial-mesenchymal transition, myogenesis, coagulation, KRAS signaling, and apical junction pathways .

  • In mesothelioma, activin B (INHBB homodimer) promotes cell invasion and migration by activating the ERK/Smad2/3 pathway .

• Cell adhesion and migration mechanisms:

  • Knockdown of INHBB increases cellular adhesion while decreasing invasiveness and migration, indicating its role in regulating these processes .

  • Treatment with recombinant activin B protein reverses these effects, confirming INHBB's direct involvement in these cellular behaviors .

These pathway insights demonstrate how INHBB antibodies serve as valuable tools for dissecting the complex molecular interactions in tumor microenvironments.

How can researchers effectively use INHBB antibodies to study epithelial-mesenchymal transition (EMT) in cancer?

INHBB antibodies offer valuable methodological approaches for studying epithelial-mesenchymal transition (EMT) in cancer:

• Co-immunostaining with EMT markers:

  • Use immunofluorescence analysis with INHBB antibodies in combination with the PathScan EMT Duplex IF Kit Primary Antibody Cocktail to visualize relationships between INHBB expression and EMT marker distribution .

  • Confocal microscopy with FluoView Software analysis enables high-resolution visualization of co-localization patterns .

• Protein expression analysis of EMT regulators:

  • Western blotting with INHBB antibodies alongside antibodies targeting EMT markers like E-cadherin, Zonula occludens-1 (Zo-1), and Snail enables quantification of their relationship .

  • This approach has been used to demonstrate how INHBB knockdown affects the expression of these EMT-related proteins .

• Functional assays coupled with INHBB manipulation:

  • Perform cellular adhesion, invasiveness, and migration assays after manipulating INHBB expression (knockdown or overexpression) .

  • Use INHBB antibodies to confirm successful manipulation of protein levels prior to functional assays .

  • Treatment with recombinant activin B can be used as a rescue experiment to confirm specificity .

• Gene expression correlation analysis:

  • Gene Set Enrichment Analysis (GSEA) has identified a significant association between INHBB and hallmark epithelial-mesenchymal transition gene sets, providing a foundation for more detailed investigations .

What emerging applications of INHBB antibodies show promise in translational research?

Several emerging applications of INHBB antibodies show significant promise for translational research:

• Liquid biopsy development: As INHBB has been shown to reflect the size of granulosa-cell tumors and can be used as a marker for primary and recurrent disease, antibodies against INHBB could potentially be developed for liquid biopsy applications to monitor cancer progression and treatment response .

• Immune checkpoint therapy biomarkers: Given INHBB's correlation with immune cell infiltration (particularly macrophages) and its relationship with immunoinhibitors/immunostimulators, INHBB antibodies may serve as prognostic or predictive biomarkers for immune checkpoint therapy response .

• Targeted therapy development: The identification of INHBB as an oncogenic driver in multiple cancers suggests that therapies targeting the activin B signaling pathway might have therapeutic potential, and INHBB antibodies would be crucial tools for developing and validating such approaches .

• Multiplexed tissue imaging: Integration of INHBB antibodies into multiplexed immunofluorescence panels would enable simultaneous visualization of INHBB expression alongside immune cell markers, providing deeper insights into the spatial relationships in the tumor microenvironment .

What are the technical limitations of current INHBB antibodies that future development should address?

Current INHBB antibodies face several technical limitations that future development efforts should address:

• Distinguishing protein complexes: Current antibodies have limited ability to differentiate between different forms of INHBB-containing complexes (inhibin B vs. activin B vs. activin AB) .

• Cross-reactivity challenges: While newer antibodies show improved specificity, further refinement is needed to eliminate any remaining cross-reactivity with related proteins .

• Application-specific optimization: Many antibodies require different protocols for different applications (IHC vs. WB vs. IF), complicating experimental design. Development of antibodies with consistent performance across applications would be valuable .

• Species-specific reactivity limitations: Due to the high conservation of INHBB across species, developing species-specific antibodies remains challenging but would be valuable for comparative studies .

• Sensitivity for low expression detection: Improved sensitivity for detecting low levels of INHBB expression would enhance research in tissues where INHBB is minimally expressed but functionally significant .

How might INHBB antibodies contribute to understanding the relationship between INHBB and cancer immunotherapy response?

INHBB antibodies could substantially advance our understanding of the relationship between INHBB and cancer immunotherapy response through several research approaches:

• Immune infiltrate characterization: Using INHBB antibodies in multiplex immunofluorescence panels to characterize the relationship between INHBB expression and immune cell infiltration patterns in patients receiving immunotherapy .

• Biomarker development: INHBB expression levels, as detected by IHC using validated antibodies, could be evaluated as potential predictive biomarkers for response to immunotherapies, particularly given INHBB's correlation with macrophage infiltration .

• Mechanistic studies: INHBB antibodies can be used to investigate how INHBB expression affects key immunomodulatory pathways. Research has already shown that INHBB expression correlates with immunoinhibitors like TGFB1 and TGFBR1, which are known to impact immunotherapy response .

• Combination therapy strategies: Understanding how INHBB influences the tumor immune microenvironment could inform combination therapy approaches, where inhibiting INHBB signaling might potentiate immunotherapy effects .

• Longitudinal monitoring: Using INHBB antibodies to monitor changes in INHBB expression during immunotherapy treatment could provide insights into resistance mechanisms and treatment dynamics .