INHBA Antibody

Basic Research Questions

• What is INHBA and what are its key functions in biological systems?

  INHBA (Inhibin subunit beta A) is a protein-coding gene belonging to the TGF-β superfamily. The protein has a molecular weight of approximately 47.4 kDa . INHBA forms a disulfide-linked homo-dimer known as activin A, which has been implicated in multiple biological processes including:

  • Regulation of follitropin secretion from the pituitary gland

  • Hypothalamic and pituitary hormone secretion

  • Gonadal hormone secretion

  • Germ cell development and maturation

  • Erythroid differentiation

  • Insulin secretion

  • Nerve cell survival

  • Embryonic axial development

  • Bone growth

  In pathological contexts, INHBA has been identified as playing critical roles in different cancer types, including esophageal squamous cell carcinoma (ESCC), head and neck squamous cell carcinoma (HNSC), gastric cancer, and colon cancer .

• What are the most common applications for INHBA antibodies in research?

  INHBA antibodies are employed in multiple experimental techniques:

  Application	Common Usage	Citation Count*
  Western Blot (WB)	Protein detection and quantification	5+ publications
  Immunohistochemistry (IHC)	Tissue localization of INHBA	11+ publications
  Immunofluorescence (IF)	Cellular localization studies	3+ publications
  ELISA	Quantitative measurement in solution	Multiple references
  Flow Cytometry (FC)	Cell-specific expression analysis	Referenced in specifications
  Knockout/Knockdown Validation	Confirming antibody specificity	1+ publications

  *Based on publication data from antibody suppliers

  Methodological approach: When selecting an INHBA antibody, researchers should match the application with validated antibodies specifically tested in that application, as antibody performance can vary significantly between techniques.

• What sample types can be effectively analyzed using INHBA antibodies?

  INHBA antibodies have demonstrated reactivity with various sample types:

  • Human tissues (most commonly validated)

  • Mouse tissues and cell lines

  • Rat specimens

  • Other mammalian species (sheep, pig, bovine, rabbit)

  INHBA antibodies have been successfully used in:

  Tissue/Organ	Research Publications
  Ovary	>22 publications
  Bone	>15 publications
  Blood	>13 publications
  Lung	>6 publications
  Skin	>5 publications
  Kidney	>5 publications
  Brain	>5 publications
  Stomach	>5 publications
  Placenta	>5 publications
  Liver	>4 publications

  Methodological approach: For optimal results, researchers should: (1) verify species reactivity before purchase, (2) use positive control tissues known to express INHBA, and (3) include appropriate blocking steps to minimize non-specific binding.

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

  Based on manufacturer recommendations:

  • Long-term storage: Maintain at -20°C to -70°C

  • Short-term storage (up to 1 month): 2-8°C under sterile conditions after reconstitution

  • Medium-term storage (up to 6 months): -20°C to -70°C under sterile conditions after reconstitution

  • Avoid repeated freeze-thaw cycles by preparing small aliquots upon initial thawing

  • Some formulations contain preservatives such as sodium azide (0.02-0.09%) and may include stabilizers like glycerol (50%)

  Methodological approach: Upon receiving an INHBA antibody, researchers should immediately divide the stock into small working aliquots before freezing to prevent protein degradation from repeated freeze-thaw cycles.

• What dilutions are typically recommended for INHBA antibodies in different applications?

  Optimal dilutions vary by application and specific antibody:

  Application	Typical Dilution Range	Notes
  Western Blot	1:1000	May observe bands at ~42-47 kDa
  IHC-P	1:10-1:50	May require antigen retrieval
  Immunofluorescence	1:10-1:50	Often requires optimization
  Flow Cytometry	1:10-1:50	Permeabilization needed for intracellular staining
  ELISA	Varies by kit	Often used as detection antibody

  Methodological approach: Always perform a dilution series experiment when using a new antibody lot or in a new experimental system. The optimal dilution may need adjustment based on expression levels in your specific samples.

Advanced Research Questions

• How can researchers validate the specificity of an INHBA antibody for their experimental system?

  Comprehensive validation approaches include:

  1. Knockout/Knockdown Controls: Compare staining between INHBA-expressing and INHBA-deficient samples. Several publications document the use of INHBA knockdown/knockout for antibody validation .

  2. Peptide Competition Assay: Pre-incubating the antibody with excess immunizing peptide should abolish specific signal.

  3. Cross-reactivity Testing: Verify specificity against related family members (particularly INHBB) to ensure the antibody discriminates between related proteins.

  4. Multi-antibody Concordance: Compare staining patterns using antibodies targeting different epitopes of INHBA.

  5. Correlation with mRNA Expression: Verify that protein expression patterns detected by the antibody correlate with mRNA expression data.

  Methodological approach: Generate INHBA-knockdown cell lines using siRNA or CRISPR-Cas9 technology for your specific cell type, then compare antibody staining between wild-type and knockdown samples. A specific antibody should show significantly reduced signal in knockdown samples.

• What are the key considerations when using INHBA antibodies in cancer research?

  INHBA has been implicated in multiple cancer types with context-dependent roles:

  Cancer Type	INHBA Association	Key Findings
  Esophageal SCC	Poor prognosis	Higher expression correlates with N categories (p=0.026) and worse OS and DFS
  Head & Neck SCC	Poor prognosis	Expression correlates with sex, TNM stage, histological grade, and HPV status
  Gastric Cancer	Unfavorable prognosis	Positively correlated with CD8+ T cells, neutrophils, macrophages, and dendritic cells
  Melanoma	Therapy resistance	Upregulated in patients resistant to immune checkpoint blockade therapy
  Colon Cancer	Promotes tumor progression	Enhances proliferation, migration and invasion of cancer cells

  Methodological considerations:

  1. Heterogeneity: INHBA expression can vary within tumor regions; consider using tissue microarrays to assess multiple regions.

  2. Subcellular Localization: INHBA expression is predominantly cytoplasmic in ESCC specimens , requiring appropriate permeabilization protocols.

  3. Control Selection: Include both tumor-adjacent normal tissue and true normal tissue as controls.

  4. Clinical Correlation: Correlate INHBA expression with clinicopathological parameters and survival data.

• How does INHBA expression correlate with immune cell infiltration and what implications does this have for antibody-based studies?

  Recent research has revealed important relationships between INHBA and tumor immunity:

  • INHBA shows negative correlation with B cell infiltration but positive correlation with macrophage infiltration in gastric cancer

  • INHBA expression positively correlates with markers of CD8+ T cells, neutrophils, macrophages, and dendritic cells

  • Tumor INHBA overexpression can promote Th2 differentiation and Tr1 infiltration while inhibiting CD8+ T cell cytotoxicity

  • INHBA effectively suppresses T cell-mediated immunity

  Methodological implications:

  1. Multiplex Staining: Consider using multiplex IHC or IF approaches to simultaneously detect INHBA and immune cell markers in the same tissue section.

  2. Spatial Analysis: Analyze the spatial relationship between INHBA-expressing cells and immune infiltrates.

  3. Functional Validation: Combine antibody-based detection with functional assays to assess how INHBA affects immune cell function.

  4. Single-cell Analysis: Consider using methods like imaging mass cytometry to correlate INHBA expression with immune cell phenotypes at single-cell resolution.

• How do researchers effectively use INHBA antibodies to study its role in resistance to cancer immunotherapy?

  INHBA has been identified as a factor in immunotherapy resistance:

  • INHBA is upregulated in melanoma patients who are resistant to immune checkpoint blockade therapy (ICB)

  • INHBA overexpression eliminates the anti-tumor function of atezolizumab (anti-PD-L1 therapy), while INHBA deficiency enhances its efficacy

  • INHBA may suppress tumor PD-L1 induced by IFN-γ from T cells, potentially leading to failure of PD-L1 blockade

  Methodological approach for studying this phenomenon:

  1. Paired Pre/Post-treatment Samples: Use INHBA antibodies to compare expression in patient samples before and after immunotherapy.

  2. Co-expression Analysis: Perform dual staining for INHBA and PD-L1 to analyze their relationship.

  3. In vitro Modeling: Use INHBA antibodies to monitor expression changes in cell lines treated with IFN-γ with or without INHBA overexpression/knockdown.

  4. Animal Models: Combine INHBA antibody staining with in vivo models of immunotherapy response to correlate expression with treatment outcomes.

  5. Mechanistic Studies: Use blocking antibodies against INHBA to determine if neutralizing INHBA can restore sensitivity to immunotherapy.

• What methodological approaches can help distinguish between different forms of INHBA (activin A vs. inhibin) in research?

  INHBA can form different protein complexes that have distinct biological functions:

  • As a homodimer (βA-βA): Forms activin A

  • Combined with inhibin α-subunit: Forms inhibin A

  • Combined with inhibin βB: Forms activin AB

  Methodological approaches to distinguish these forms:

  1. Epitope-specific Antibodies: Use antibodies that specifically recognize epitopes only accessible in certain configurations.

  2. Sequential Immunoprecipitation: First precipitate with an antibody against one subunit, then probe the precipitate with antibodies against potential partner subunits.

  3. Non-reducing vs. Reducing Western Blots: Under non-reducing conditions, observe the intact dimers; under reducing conditions, observe individual subunits.

  4. Size Exclusion Chromatography: Separate different complexes based on molecular weight before antibody detection.

  5. Functional Assays: Complement antibody detection with bioassays that distinguish the different biological activities of activins versus inhibins.

• How can INHBA antibodies be leveraged in developing potential therapeutic approaches for cancer?

  Given INHBA's role in cancer progression and therapy resistance, antibody-based approaches have research applications:

  1. Target Validation: Use antibodies to confirm INHBA as a therapeutic target through localization and interaction studies.

  2. Biomarker Development: INHBA has been identified as a potential biomarker for:

     • Anti-TGFβ therapy in HNSC

     • Predictor of immunotherapy response in gastric cancer

     • Prognostic indicator in multiple cancer types

  3. Neutralizing Antibody Development: Research antibodies can inform the development of therapeutic antibodies that neutralize INHBA/Activin A.

  4. Combination Therapy Assessment: Use INHBA antibodies to monitor expression changes during various treatments to identify potential synergistic approaches.

  Methodological strategy: For biomarker development, researchers should establish standardized IHC protocols for INHBA detection and define clear scoring criteria that correlate with clinical outcomes.