TGFB1 Human
Description
The TGF-b 1 is purified by proprietary chromatographic techniques.
Product Specs
Q&A
How do researchers quantify TGFB1 expression in human tissue samples?
TGFB1 expression analysis requires validation at transcriptional and translational levels:
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qPCR Methodology: Use primers targeting exonic regions (e.g., TGFB1 NM_000660.6) with reference genes like GAPDH. Amplification efficiency must be calculated via standard curves (e.g., 101% efficiency for TGFB1 vs. 111% for GAPDH), necessitating Pfaffl’s method for relative quantification .
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Protein Detection: ELISA with antibodies specific for latent (LAP-TGFB1) or active forms. Note that latent complexes dominate extracellular matrices (ECMs), requiring acid/alkaline activation for accurate measurement .
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Troubleshooting: Cross-validate with immunohistochemistry to confirm cellular localization, as stromal vs. tumor cell expression impacts biological interpretation .
What experimental models elucidate TGFB1’s role in disease progression?
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In Vitro Systems: Primary fibroblasts or cancer cell lines (e.g., HT-29 for colorectal cancer) treated with recombinant TGFB1 (2–10 ng/mL) to assess EMT (Epithelial-Mesenchymal Transition) markers (e.g., α-SMA, fibronectin) .
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Animal Models: Conditional Tgfb1 knockout mice with tissue-specific Cre drivers (e.g., Col1a2-Cre for fibrosis studies). Monitor compensatory TGFB isoform upregulation .
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Clinical Cohorts: Retrospective analysis of TCGA datasets to correlate TGFB1 mRNA levels with survival outcomes in hematological malignancies .
How does TGFB1 regulate immune responses in cancer microenvironments?
TGFB1 exhibits dual pro- and anti-tumor roles depending on context:
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Immunosuppression: Upregulates regulatory T cells (Tregs) and inhibits CD8+ T cell cytotoxicity via SMAD3-dependent pathways. Assess using flow cytometry of tumor-infiltrating lymphocytes (TILs) .
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Stromal Remodeling: Induces fibroblast-to-myofibroblast differentiation, increasing ECM stiffness. Measure via Atomic Force Microscopy (AFM) in 3D collagen matrices .
Table 1: TGFB1 Expression and Prognosis in Hematological Cancers
What epigenetic mechanisms govern TGFB1 activity?
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DNA Methylation: Methylation-specific PCR (MS-PCR) of CpG islands in the TGFB1 promoter (e.g., chr19:41,834,318–41,834,744). Use bisulfite conversion and primers distinguishing methylated/unmethylated sequences .
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Histone Modifications: ChIP-seq for H3K27ac or H3K4me3 marks at enhancer regions. Correlate with RNA-seq data to identify transcriptionally active loci .
Key Finding:
In colorectal cancer, TGFB1 promoter methylation inversely correlates with mRNA levels (r = -0.62, p < 0.01), but no association with age or histologic subtype was observed .
How are TGFB1 genetic polymorphisms analyzed for disease associations?
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SNP Selection: Focus on functional variants (e.g., rs1800470 (codon 10) and rs1800471 (codon 25)) linked to altered TGF-β1 secretion. Genotype via TaqMan assays .
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Statistical Models: Apply additive, dominant, and recessive models using PLINK. For meta-analyses, use fixed-effects models if heterogeneity is low (I² < 25%) .
Table 2: Meta-Analysis of TGFB1 Codon 10/25 Polymorphisms in Chronic Allograft Dysfunction
| Polymorphism | Genetic Model | Odds Ratio (95% CI) | p-Value |
|---|---|---|---|
| Codon 10 (T/C) | Dominant | 1.37 (0.61–3.06) | 0.44 |
| Codon 25 (G/C) | Allelic | 1.12 (0.82–1.53) | 0.47 |
How do researchers resolve contradictions in TGFB1’s context-dependent roles in cancer?
Experimental Design:
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Stratified Analysis: Subgroup tumors by mutational burden (e.g., TP53 status) or microenvironmental features (e.g., fibroblast density). Single-cell RNA-seq can deconvolute cell-type-specific TGFB1 expression .
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Pharmacologic Inhibition: Treat patient-derived organoids with galunisertib (TGFBR1 inhibitor) and monitor changes in metastatic potential vs. immune evasion .
Case Study: In DLBCL, stromal TGFB1 correlates with immune exclusion, whereas tumor-intrinsic expression associates with PD-L1 upregulation .
What unconventional secretion pathways transport TGFB1, and how are they studied?
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Secretory Autophagy: Knockdown ATG5 or RAB8A in fibroblasts to block LC3+ autophagosome formation. Confirm via immunogold TEM and Western blot for LAP-TGFB1 in extracellular vesicles .
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Live-Cell Imaging: Tag latent TGFB1 with pH-sensitive fluorescent probes (e.g., pHluorin) to track release from recycling endosomes .
Technical Challenge:
Differentiating constitutive secretion (≈30% of total TGFB1) from stress-induced unconventional pathways requires pulse-chase assays with 35S-methionine labeling .
How can conflicting findings about TGFB1’s prognostic value be reconciled?
Data Triangulation Framework:
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Cohort Stratification: Validate in independent datasets (e.g., GEO: GSE135222 for AML) pre-stratified by molecular subtype.
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Multivariate Modeling: Adjust for covariates like age, stage, and treatment history. In TCGA-LAML, TGFB1 remains prognostic after adjusting for cytogenetic risk (HR = 1.4, p = 0.03) .
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Mechanistic Studies: Use CRISPRa to overexpress TGFB1 in isogenic cell lines and assess metastatic potential in zebrafish xenografts.
What methodologies identify TGFB1 isoform-specific functions?
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Isoform-Specific Knockdown: Design siRNAs targeting unique 3’UTR regions of TGFB1 (NM_000660) vs. TGFB2 (NM_003238). Confirm specificity via qPCR and Luminex multiplex assays.
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Structural Biology: Solve cryo-EM structures of TGFB1-LAP complexes bound to integrins (e.g., αvβ6) to map activation interfaces .
How is TGFB1 integrated into multi-omics frameworks for therapeutic discovery?
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Network Analysis: Build protein-protein interaction networks using STRING-DB, highlighting crosstalk with PD-1/PD-L1 and VEGF pathways.
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Machine Learning: Train random forest models on TCGA data using TGFB1, immune scores, and mutation count to predict immunotherapy response (AUC = 0.78) .
Table 3: Predictive Value of TGFB1 in Immunotherapy Response
| Biomarker | Cancer Type | AUC | Sensitivity | Specificity |
|---|---|---|---|---|
| Serum TGFB1 | Melanoma | 0.72 | 68% | 81% |
| Tumor TGFB1 mRNA | NSCLC | 0.65 | 57% | 76% |
Methodological Recommendations
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Sample Size Justification: For SNP studies, use Quanto to calculate power based on minor allele frequency (MAF > 0.2) and expected effect size (OR > 2.0) .
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Replication Cohorts: Cross-validate findings in ≥2 independent populations (e.g., Hemap and GTEx) .
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Data Transparency: Share raw qPCR Ct values and electrophoresis images via repositories like Figshare.
Product Science Overview
TGF-β1 is a multifunctional cytokine that performs several key functions:
- Cell Growth and Proliferation: TGF-β1 regulates the growth and proliferation of various cell types. It acts synergistically with transforming growth factor-alpha (TGF-α) in inducing cellular transformation .
- Immune System Regulation: TGF-β1 plays a significant role in controlling the immune system. It is secreted by most immune cells and can inhibit the actions of other cytokines, such as interleukin-1 (IL-1) and interleukin-2 (IL-2), thereby modulating immune responses .
- Wound Healing: TGF-β1 was first identified in human platelets and has a potential role in wound healing due to its ability to control cell growth and differentiation .
Dysregulation of TGF-β1 activation and signaling can lead to various diseases:
- Cancer: TGF-β1 acts as a tumor suppressor by inhibiting cell proliferation and inducing apoptosis. However, in tumor cells, TGF-β1 can lose its anti-proliferative response and become an oncogenic factor, promoting tumor progression .
- Fibrosis and Inflammation: Chronic overexpression of TGF-β1 is associated with diseases such as fibrosis and inflammation. It drives disease progression by modulating cell growth and migration .
