Recombinant Human Inhibin beta A chain (INHBA) (Active)

What is Recombinant Human Inhibin beta A chain (INHBA) and what are its primary biological functions?

Recombinant Human Inhibin beta A chain (INHBA) is a protein belonging to the Transforming Growth Factor-beta (TGF-beta) superfamily. The active form corresponds to amino acids 311-426 of the full-length protein and is typically expressed in systems like Escherichia coli or mammalian cells with >90-95% purity .

Its primary biological functions include regulating follitropin secretion from the pituitary gland, with inhibins inhibiting and activins activating this process. INHBA participates in numerous physiological processes including:

• Hypothalamic and pituitary hormone secretion regulation

• Gonadal hormone secretion control

• Germ cell development and maturation

• Erythroid differentiation

• Insulin secretion modulation

• Neural cell survival promotion

• Embryonic axial development

• Bone growth regulation

The protein's activities are highly context-dependent, often determined by its dimerization state and interaction with specific receptors in target tissues.

How do I determine the appropriate concentration of Recombinant Human Inhibin beta A for my experiments?

Determining the appropriate concentration of Recombinant Human Inhibin beta A for experimental use depends on your specific research application, cell type, and desired biological effect. Generally, effective concentration ranges have been established through empirical testing:

For activin-mediated effects on follicle-stimulating hormone (FSH) release, the effective concentration (ED50) typically ranges from 0.3-1.5 ng/mL . This concentration range is often suitable as a starting point for experiments investigating pituitary hormone regulation.

For cell culture applications investigating cellular responses:

• For proliferation assays: 1-20 ng/mL

• For differentiation studies: 5-50 ng/mL

• For receptor binding studies: 10-100 ng/mL

It is recommended to perform a dose-response experiment to determine the optimal concentration for your specific experimental system. Start with a range spanning from 0.1 ng/mL to 100 ng/mL and narrow down based on observed effects. Positive controls using well-established activin-responsive cell lines can help validate activity.

The biological potency of commercial preparations may vary between manufacturers, so it's essential to refer to the specific activity information provided with your recombinant protein .

What are the optimal storage and handling conditions for Recombinant Human Inhibin beta A to maintain its activity?

Proper storage and handling of Recombinant Human Inhibin beta A is crucial to preserve its biological activity. Based on manufacturer recommendations and research protocols, the following practices should be observed:

Storage conditions:

• Store lyophilized protein at -20°C to -80°C for long-term storage

• Once reconstituted, aliquot the protein to avoid repeated freeze-thaw cycles

• Store reconstituted protein at -80°C for up to 3-6 months

• Avoid more than 2-3 freeze-thaw cycles as protein activity may diminish with each cycle

Reconstitution protocols:

• Reconstitute in sterile, buffer solutions such as PBS or appropriate cell culture medium

• For higher concentration stocks, reconstitution in 4 mM HCl containing 0.1% human serum albumin (HSA) or bovine serum albumin (BSA) as a carrier protein can help stabilize the protein

• Filter through a 0.22 μm filter if needed for sterility

• Allow the protein to sit for at least 10 minutes at room temperature after adding reconstitution buffer to ensure complete solubilization

Working solutions:

• Prepare working solutions on ice

• Use polypropylene tubes to prevent protein adherence to container walls

• When diluting to working concentrations, use medium containing carrier proteins (0.1-0.5% BSA) to prevent loss of activity due to adsorption to labware

Handling precautions:

• Avoid vigorous vortexing which can denature the protein

• Use gentle pipetting techniques when preparing dilutions

• Maintain endotoxin levels below 1 EU/μg for cell culture applications

How can I validate the biological activity of Recombinant Human Inhibin beta A in my experimental system?

Validating the biological activity of Recombinant Human Inhibin beta A is essential to ensure experimental reproducibility. Several established methods can be used:

1. Bioassay using follicle-stimulating hormone (FSH) secretion:

• Cultured pituitary cells or appropriate pituitary cell lines can be treated with the recombinant protein

• Measure FSH secretion using ELISA or radioimmunoassay

• Active Inhibin beta A (as part of Activin) will show an ED50 of approximately 0.3-1.5 ng/mL

2. SMAD phosphorylation assay:

• As a member of the TGF-beta family, Inhibin beta A activates SMAD signaling pathways

• Treat responsive cells with the recombinant protein for 30-60 minutes

• Use Western blotting to detect phosphorylation of SMAD2/3 proteins

• Include positive controls such as TGF-beta1

3. Reporter gene assays:

• Utilize cells transfected with SMAD-responsive elements coupled to a reporter gene (luciferase)

• Treatment with active Inhibin beta A will induce reporter gene expression

• Quantify luminescence as a measure of signaling activation

4. Receptor binding assays:

• Surface plasmon resonance (SPR) or similar techniques can be used to measure direct binding to activin receptors

• Radiolabeled or fluorescently labeled protein can be used in competitive binding assays

5. Functional cell-based assays:

• For erythroid differentiation: measure hemoglobin production in K562 cells

• For stem cell studies: assess changes in pluripotency markers in embryonic stem cells

• For reproductive biology: measure effects on granulosa cell proliferation or steroidogenesis

A positive validation should include both dose-dependent responses and specificity controls (such as neutralizing antibodies against Inhibin beta A or its receptors).

What are the most effective methods for studying Inhibin beta A interactions with its receptors?

Studying the interactions between Inhibin beta A and its receptors requires specialized techniques that can detect binding events and subsequent signaling processes:

1. Surface Plasmon Resonance (SPR):

• Immobilize purified activin receptors (ActRII-A, ActRII-B, or ActRI-B) on sensor chips

• Flow Recombinant Human Inhibin beta A over the surface

• Measure real-time binding kinetics, including association and dissociation rates

• Determine binding affinity (KD) values

• This technique is particularly valuable for comparing binding properties across different receptor subtypes

2. Co-immunoprecipitation (Co-IP):

• Express tagged versions of receptors in cell lines

• Treat with Recombinant Human Inhibin beta A

• Precipitate receptor complexes using antibodies

• Analyze complex formation by Western blotting

• This approach reveals natural complex formation in cellular contexts

3. FRET/BRET-based approaches:

• Generate fluorescently tagged Inhibin beta A and receptor constructs

• Co-express in suitable cell lines

• Measure fluorescence or bioluminescence resonance energy transfer

• This provides real-time, live-cell analysis of protein-protein interactions

4. Receptor crosslinking studies:

• Radiolabel Recombinant Human Inhibin beta A (e.g., with 125I)

• Incubate with cells expressing activin receptors

• Use chemical crosslinkers to stabilize the interactions

• Identify receptor complexes by autoradiography after SDS-PAGE

• This technique can reveal the composition of receptor complexes

5. Signaling cascade analysis:

• Monitor downstream signaling events after receptor binding

• Focus on SMAD2/3 phosphorylation as primary mediators

• Also examine non-canonical pathways (MAPK, PI3K/AKT)

• Use small molecule inhibitors and dominant-negative constructs to validate specificity

6. Cryo-electron microscopy (Cryo-EM):

• For structural studies of the ligand-receptor complex

• Provides atomic-level details of interaction interfaces

• Can reveal conformational changes upon binding

A comprehensive approach would combine several of these methods to build a complete picture of Inhibin beta A-receptor interactions.

How do post-translational modifications affect the function of Inhibin beta A?

Post-translational modifications (PTMs) of Inhibin beta A significantly influence its biological activity, processing, and receptor interactions. Understanding these modifications is crucial for interpreting experimental results:

1. Glycosylation:

• Native Inhibin beta A contains N-linked glycosylation sites

• Recombinant protein from E. coli lacks glycosylation, while mammalian cell-produced proteins maintain these modifications

• Glycosylation affects:

  • Protein stability and half-life in circulation

  • Receptor binding affinity

  • Susceptibility to proteolytic degradation

  • Tissue distribution and clearance rates

2. Proteolytic processing:

• Inhibin beta A is synthesized as a precursor protein

• Proteolytic cleavage by proprotein convertases (e.g., furin) is required to release the mature, active domain

• The timing and efficiency of this processing regulates active protein availability

• Mutations affecting cleavage sites can lead to altered bioactivity

3. Disulfide bond formation:

• The mature Inhibin beta A domain contains a characteristic pattern of disulfide bonds essential for its three-dimensional structure

• Correct disulfide pairing is critical for proper folding and receptor recognition

• Recombinant proteins must maintain these bonds for full biological activity

• Reducing agents should be avoided in experimental buffers

4. Phosphorylation:

• Certain serine/threonine residues may undergo phosphorylation

• This can modulate receptor binding properties and signaling outcomes

• Phosphorylation status may vary depending on the cellular context and physiological conditions

Experimental considerations:

• For studies requiring precise control of PTMs, consider the expression system carefully

• E. coli-produced proteins lack glycosylation but may be suitable for many functional assays

• Mammalian cell-produced proteins better represent the native glycosylation pattern

• For studies of receptor binding kinetics, the glycosylation status should be consistent across experiments

What role does Inhibin beta A play in reproductive physiology at the molecular level?

Inhibin beta A plays crucial roles in reproductive physiology through complex molecular mechanisms in both male and female reproductive systems:

Female reproductive system:

• Folliculogenesis regulation:

  • As a component of Activin A (beta A-beta A homodimer), it promotes granulosa cell proliferation

  • Enhances FSH receptor expression in granulosa cells

  • Stimulates early follicular development from primary to antral stages

  • Modulates follicle sensitivity to gonadotropins

• Oocyte maturation:

  • Promotes expansion of cumulus cells surrounding the oocyte

  • Enhances oocyte developmental competence

  • Regulates meiotic progression in oocytes

• Hypothalamic-pituitary-gonadal axis:

  • Activin A stimulates FSH synthesis and secretion from pituitary gonadotropes

  • Inhibin A (alpha-beta A heterodimer) suppresses FSH production

  • This creates a feedback loop essential for cyclic ovarian function

Male reproductive system:

• Spermatogenesis:

  • Regulates proliferation and differentiation of spermatogonia

  • Influences Sertoli cell function to support germ cell development

  • Modulates the blood-testis barrier integrity

• Steroidogenesis:

  • Affects testosterone production by Leydig cells

  • Interacts with other factors to regulate steroid hormone balance

Molecular mechanisms:

• Receptor-mediated signaling:

  • Activin A (beta A-beta A) binds preferentially to type II activin receptors (ActRII-A and ActRII-B)

  • This binding recruits and phosphorylates type I receptors (primarily ALK4)

  • Activated type I receptors phosphorylate SMAD2/3 proteins

  • Phosphorylated SMAD2/3 forms complexes with SMAD4 and translocates to the nucleus

  • These complexes regulate gene expression of targets including:

    • Cyclin D2 (proliferation)

    • FSH receptor (gonadotropin sensitivity)

    • Steroidogenic enzymes (hormone production)

• Regulation by binding proteins:

  • Follistatin binds Activin A with high affinity, neutralizing its activity

  • This interaction provides an additional layer of control over Inhibin beta A function

  • The follistatin:Inhibin beta A ratio in follicular fluid correlates with follicle health and developmental potential

Understanding these molecular mechanisms is essential for research in reproductive biology, fertility treatments, and contraceptive development.

How do Inhibin beta A dimers differ in their biological activities and signaling properties?

Inhibin beta A can form various dimeric configurations, each with distinct biological activities and signaling properties that significantly impact experimental outcomes:

Table 1: Comparison of Inhibin beta A-containing dimers

Signaling pathway variations:

• Activin A (β_A-β_A):

  • Binds with high affinity to ActRII-A/B

  • Recruits and activates ALK4 (type I receptor)

  • Triggers robust SMAD2/3 phosphorylation

  • Can also activate non-canonical pathways including MAPK and PI3K/AKT

  • Signal strength is typically stronger than other activin forms

• Activin AB (β_A-β_B):

  • Shares receptor preferences with Activin A

  • Generally shows intermediate signaling potency

  • May have tissue-specific effects distinct from Activin A

• Inhibin A (α-β_A):

  • Acts as a competitive antagonist by binding ActRII without recruiting type I receptors

  • Requires co-receptor betaglycan for high-affinity binding

  • Blocks Activin-mediated SMAD2/3 phosphorylation

  • Functions primarily as a negative regulator of activin signaling

Functional consequences in research applications:

• Receptor binding studies must account for the specific dimer being studied, as binding affinities vary significantly

• Cell-based assays will show different dose-response relationships depending on which dimer is applied

• In vivo studies may reveal tissue-specific effects based on the expression patterns of receptors and co-receptors

• Developmental biology research should consider the temporally regulated expression of different dimers during embryogenesis

The study of these different dimeric forms requires careful experimental design and appropriate controls to distinguish their specific effects. Researchers should clearly identify which specific dimeric form they are working with in their experimental protocols and data reporting.

What are common issues encountered when working with Recombinant Human Inhibin beta A and how can they be resolved?

Researchers working with Recombinant Human Inhibin beta A may encounter several challenges that can affect experimental outcomes. Here are common issues and their solutions:

1. Loss of protein activity during storage/handling:

• Issue: Repeated freeze-thaw cycles or improper storage leading to reduced biological activity

• Solution: Aliquot reconstituted protein into single-use volumes and store at -80°C. Limit freeze-thaw cycles to a maximum of 2-3. Add carrier proteins (0.1% BSA or HSA) to dilute solutions to prevent adsorption to surfaces

2. Inconsistent experimental results:

• Issue: Variation in protein activity between experiments

• Solution: Standardize reconstitution protocols and use consistent buffer compositions. Implement a quality control bioassay (e.g., SMAD phosphorylation) to verify activity before critical experiments. Use the same lot number when possible for a series of related experiments.

3. Low protein solubility:

• Issue: Precipitation or aggregation of recombinant protein

• Solution: Reconstitute in acidified buffer (4mM HCl) with carrier protein, then dilute in experimental medium. Centrifuge solutions briefly before use to remove any precipitates. Avoid buffers with high salt concentrations.

4. Endotoxin contamination:

• Issue: Endotoxin in E. coli-derived preparations affecting cell culture experiments

• Solution: Verify endotoxin levels are <1 EU/μg for cell culture applications. For sensitive experiments, consider mammalian cell-derived preparations or additional endotoxin removal steps

5. Interference with detection methods:

• Issue: Background signals in immunoassays or activity assays

• Solution: Include appropriate negative controls (heat-inactivated protein or unrelated recombinant protein). For Western blotting, use antibodies specific to the human sequence to avoid cross-reactivity.

6. Difficulty distinguishing effects of different activin/inhibin dimers:

• Issue: Overlapping biological activities between different inhibin/activin dimers

• Solution: Use specific neutralizing antibodies against Inhibin beta A. Compare with recombinant Activin B or other family members. Consider receptor-specific approaches to distinguish signaling pathways.

7. Concentration determination challenges:

• Issue: Inaccurate protein quantification

• Solution: Use multiple quantification methods (Bradford, BCA, and UV absorbance) and take the average. Standardize against known protein standards in similar buffer conditions.

8. Cell type-specific responsiveness:

• Issue: Variability in cellular responses between different cell types

• Solution: Characterize receptor expression in your cell system before experiments. Include positive control cell lines with known responsiveness to Inhibin beta A (e.g., HepG2 for SMAD signaling).

How can I distinguish between the activities of Inhibin beta A and other TGF-beta family members in my experiments?

Distinguishing between the biological activities of Inhibin beta A and other TGF-beta family members requires specific experimental approaches to ensure accurate interpretation of results:

1. Receptor utilization analysis:

• Different TGF-beta family members activate distinct receptor combinations

• Inhibin beta A (as Activin A) primarily signals through ActRII-A/B and ALK4

• Use receptor-specific inhibitors:

  • SB-431542 inhibits ALK4/5/7 (blocks Activin, TGF-β, Nodal, but not BMP signaling)

  • K02288 inhibits ALK1/2/3/6 (blocks BMP, but not Activin signaling)

• Receptor knockdown/knockout approaches using siRNA or CRISPR-Cas9 can confirm specific receptor requirements

2. Downstream signaling discrimination:

• Inhibin beta A primarily activates SMAD2/3 phosphorylation

• BMPs predominantly activate SMAD1/5/8

• TGF-β activates both SMAD2/3 and non-SMAD pathways

• Western blotting for phosphorylated SMAD proteins can identify pathway specificity

• Time-course studies can distinguish between early and late signaling events

3. Specific neutralizing antibodies:

• Use antibodies specifically recognizing Inhibin beta A

• Compare with antibodies targeting other TGF-beta family members

• Validation should include dose-dependent neutralization of known activities

4. Competitive binding assays:

• Pre-incubate cells with excess unlabeled ligands

• Challenge with labeled or tagged Inhibin beta A

• Displacement patterns can reveal binding specificity

5. Gene expression profiling:

• Different TGF-beta family members induce distinct transcriptional signatures

• RNA-seq or qPCR of known target genes can distinguish specific responses:

  • Inhibin beta A/Activin A strongly induces SMAD7, SERPINE1, JUNB

  • BMPs strongly induce ID1, ID2, ID3

  • TGF-β strongly induces CTGF, COL1A1, FN1

Experimental approach table:

By combining several of these approaches, researchers can confidently attribute observed effects to Inhibin beta A rather than other TGF-beta family members.

How is Recombinant Human Inhibin beta A being used in stem cell research and regenerative medicine?

Recombinant Human Inhibin beta A, particularly as part of Activin A (beta A-beta A homodimer), has become a critical tool in stem cell research and regenerative medicine applications:

1. Pluripotent stem cell maintenance and differentiation:

• Activin A supports the self-renewal of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) by activating SMAD2/3 signaling

• Typical working concentrations range from 10-50 ng/mL for maintenance protocols

• When used in defined media formulations, it can replace the need for feeder cells or undefined supplements

• Precise temporal modulation of Activin A signaling directs differentiation toward specific lineages:

  • Sustained high levels (50-100 ng/mL) promote definitive endoderm formation

  • Transient exposure followed by inhibition facilitates mesoderm induction

  • Inhibition promotes neuroectoderm differentiation

2. Organoid development:

• Activin A is a key component in protocols generating various organoids:

  • Intestinal organoids: Used at 50-100 ng/mL during initial endoderm specification

  • Liver organoids: Critical for hepatocyte maturation at 10-20 ng/mL

  • Pancreatic organoids: Used at defined concentrations during pancreatic progenitor induction

3. Reproductive medicine applications:

• Recombinant Inhibin beta A is used to study follicle development in vitro

• It contributes to improved in vitro maturation protocols for oocytes

• Helps develop better culture systems for primordial follicle activation and growth

• May have applications in fertility preservation technologies

4. Modeling developmental processes:

• Used to recapitulate embryonic patterning in vitro

• Helps establish anterior-posterior axis formation in gastruloid models

• Concentration gradients of Activin A can establish developmental territories similar to those in early embryos

5. Disease modeling with iPSCs:

• Patient-derived iPSCs treated with defined Activin A protocols can generate disease-relevant cell types

• Enables the study of developmental disorders related to TGF-beta signaling pathways

• Particularly valuable for reproductive, endocrine, and developmental disorder research

6. Bioengineering approaches:

• Controlled release systems incorporating Activin A improve directed differentiation

• Biomaterial scaffolds with immobilized Activin A enhance spatial control of stem cell fate

• Microfluidic devices creating Activin A gradients better mimic developmental environments

The applications of Recombinant Human Inhibin beta A continue to expand as our understanding of its roles in development and cellular differentiation deepens. Researchers working with stem cells should carefully optimize concentration, timing, and combinatorial factor approaches for their specific experimental systems.

What are the latest advances in using CRISPR/Cas9 technology to study Inhibin beta A function?

CRISPR/Cas9 technology has revolutionized the study of Inhibin beta A function, enabling precise genetic manipulation approaches that were previously challenging or impossible. Recent advances in this area include:

1. Genomic editing of the INHBA gene:

• Knockout models: Complete elimination of Inhibin beta A expression to study loss-of-function phenotypes

• Knockin approaches: Introduction of reporter genes (GFP, luciferase) to track endogenous expression patterns

• Point mutations: Creation of specific amino acid substitutions to study structure-function relationships

• These approaches have revealed previously unknown roles in diverse processes including inflammation, wound healing, and cancer progression

2. Regulation of INHBA expression:

• Promoter editing: Modification of regulatory regions to alter expression levels

• CRISPR interference (CRISPRi): Targeted repression of INHBA transcription

• CRISPR activation (CRISPRa): Upregulation of endogenous INHBA expression

• These strategies provide more physiologically relevant models than exogenous protein addition

3. Receptor interaction studies:

• Mutation of specific receptor binding domains in the INHBA gene

• Creation of chimeric ligands to study domain-specific functions

• Engineering of altered binding specificities

• These approaches help distinguish the specific contributions of different receptor subtypes to Inhibin beta A signaling

4. Lineage tracing and developmental studies:

• CRISPR-mediated insertion of Cre-recombinase under INHBA promoter control

• Temporal control using inducible Cas9 systems to study stage-specific functions

• These techniques have revealed previously unknown sources of Inhibin beta A during development

5. High-throughput screening approaches:

• CRISPR libraries targeting genes in the Inhibin beta A signaling pathway

• Identification of novel regulators and effectors

• These screens have uncovered unexpected interactions with other signaling networks

6. Therapeutic potential exploration:

• Correction of INHBA mutations associated with developmental disorders

• Modulation of Inhibin beta A expression in disease models

• These studies suggest potential for gene therapy approaches in conditions with dysregulated Inhibin beta A signaling

7. Advanced delivery systems:

• Tissue-specific CRISPR delivery to manipulate INHBA in select cell populations

• Temporal control using optogenetic or chemically inducible Cas9 variants

• These approaches minimize developmental compensation that can confound conventional knockout studies

Methodological considerations for CRISPR/Cas9 studies of INHBA:

• Design multiple gRNAs targeting different exons to ensure complete knockout

• Validate editing efficiency using both genomic sequencing and protein expression analysis

• Include rescue experiments with recombinant protein to confirm specificity

• Consider potential compensatory upregulation of related family members (e.g., Inhibin beta B)

These advanced CRISPR/Cas9 approaches have significantly expanded our understanding of Inhibin beta A biology beyond what was possible with conventional techniques.

What are the most important considerations when designing experiments involving Recombinant Human Inhibin beta A?

When designing experiments involving Recombinant Human Inhibin beta A, researchers should consider several critical factors to ensure reliable and interpretable results:

1. Protein selection and quality:

• Choose between E. coli-expressed (~90-95% purity) and mammalian cell-expressed preparations based on your experimental needs

• Verify activity before use, particularly for critical experiments

• Consider the absence or presence of post-translational modifications as relevant to your research question

• Use preparations with certified low endotoxin levels (<1 EU/μg) for cell culture applications

2. Dimerization state awareness:

• Clearly distinguish which form you are investigating: Activin A (βA-βA), Activin AB (βA-βB), or Inhibin A (α-βA)

• These different dimeric forms have distinct receptor affinities and biological activities

• Commercial preparations may contain specific dimeric forms, so verify product specifications

3. Dosage and timing considerations:

• Establish dose-response relationships for your specific cell type or tissue

• Consider that effective concentrations range from 0.3-100 ng/mL depending on the biological response being measured

• Include time-course studies as responses may vary from rapid (minutes for SMAD phosphorylation) to delayed (hours/days for differentiation)

• Be aware that sustained vs. pulsatile exposure may produce different outcomes

4. Appropriate controls:

• Include both positive controls (known responsive systems) and negative controls (heat-inactivated protein)

• Consider using neutralizing antibodies or receptor antagonists as specificity controls

• For comparative studies with other TGF-beta family members, ensure equivalent molar concentrations rather than weight/volume

5. Cell/tissue context awareness:

• Verify receptor expression in your experimental system

• Consider the presence of endogenous inhibitors (follistatin, noggin) or co-receptors (betaglycan)

• Account for potential autocrine/paracrine production of related ligands

6. Downstream analysis selection:

• Choose assays appropriate for the expected response (e.g., SMAD phosphorylation for immediate signaling, gene expression for longer-term effects)

• Consider pathway crosstalk in data interpretation

• Validate key findings using complementary techniques

7. Experimental design rigor:

• Include biological replicates (different cell preparations) and technical replicates

• Design experiments with appropriate statistical power

• Pre-register hypotheses and analysis plans when possible

• Document lot numbers and sources of recombinant proteins used

By carefully considering these factors, researchers can design robust experiments that maximize the reliability and reproducibility of their findings involving Recombinant Human Inhibin beta A.

How can researchers integrate multi-omics approaches to better understand Inhibin beta A signaling networks?

Integrating multi-omics approaches provides a comprehensive understanding of Inhibin beta A signaling networks beyond what single-technique studies can reveal. This systems biology approach enables researchers to map complex cellular responses across multiple levels of biological organization:

1. Transcriptomics integration:

• RNA-seq following Inhibin beta A treatment identifies direct and indirect target genes

• Time-course studies distinguish early vs. late response genes

• Single-cell RNA-seq reveals cell-specific responses within heterogeneous populations

• ATAC-seq identifies changes in chromatin accessibility at regulatory regions

• ChIP-seq for SMAD2/3 maps direct binding sites and identifies DNA motifs

• Integration of these data creates temporal transcriptional regulatory networks

2. Proteomics contributions:

• Phosphoproteomics captures immediate signaling events (minutes to hours)

• Quantitative proteomics reveals changes in protein abundance (hours to days)

• Proximity labeling techniques (BioID, APEX) identify context-specific protein interactions

• Cross-linking mass spectrometry detects direct binding partners

• Integration with transcriptomics highlights post-transcriptional regulation mechanisms

3. Metabolomics insights:

• Targeted and untargeted metabolomics reveal changes in cellular metabolism

• Stable isotope labeling tracks metabolic flux alterations

• Integration with proteomics identifies enzyme activity changes not apparent from expression data

• Particular relevance for understanding Inhibin beta A effects on energy metabolism and steroidogenesis

4. Computational integration frameworks:

• Network analysis identifies hub genes/proteins in Inhibin beta A response networks

• Pathway enrichment across multiple omics layers reveals consistent biological processes

• Machine learning approaches predict new regulatory connections

• Causal network inference distinguishes drivers from passengers in signaling cascades

5. Validation and functional characterization:

• CRISPR screens validate predicted network components

• Perturbation studies confirm computational predictions

• Real-time biosensors monitor pathway activity in living cells

• These experimental approaches close the loop between prediction and validation

Recommended multi-omics experimental design: