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Tgfbr1

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TGFBR1 is a protein-coding gene that encodes the transforming growth factor beta receptor type-1. This receptor mediates the signal of various cytokines of the TGF beta superfamily and is involved in the regulation of cellular processes such as cell growth, cell differentiation, and apoptosis.

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Lab products found in correlation

Smad3, by Cell Signaling Technology (3 mentions) Tgfbr2, by Cell Signaling Technology (2 mentions) Tgfbr2, by Santa Cruz Biotechnology (2 mentions) Vimentin, by Santa Cruz Biotechnology (2 mentions) N cadherin, by BD (2 mentions) Fibronectin, by Santa Cruz Biotechnology (2 mentions) Cleaved, by Cell Signaling Technology (1 mentions) Phospho smad3, by Cell Signaling Technology (1 mentions) Hsp90, by Santa Cruz Biotechnology (1 mentions) Tsp 1, by Santa Cruz Biotechnology (1 mentions) Hsp90, by Bio-Rad (1 mentions) Amicon ultra centrifugal filter, by Merck Group (1 mentions) α sma, by Merck Group (1 mentions) β actin, by Santa Cruz Biotechnology (1 mentions) Chemidoc touch imaging system, by Bio-Rad (1 mentions) Image lab software v5, by Bio-Rad (1 mentions) Enhanced chemiluminescence solution, by Cytiva (1 mentions) Western blotting detection reagent, by GE Healthcare (1 mentions) Nitrocellulose membrane, by Cytiva (1 mentions) Fgfr1, by Santa Cruz Biotechnology (1 mentions)

10 protocols using tgfbr1

1

Analyzing Protein Expression in Cell Lysates

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Protein was extracted from cell or tissue lysates and analyzed using SDS-PAGE.49 (link) Antibodies used were α-SMA (Sigma-Aldrich, MERCK, Singapore), cleaved caspase-3, phospho-SMAD3 (Ser423/425), SMAD3, phospho-JNK1 (Thr183/Tyr185), JNK1, TGFbr2 (Cell Signaling Technology, Research Biolabs, Singapore), BAK1, P53, TSP1, ETS1, TGFbr1, β-actin, HSP90 (Santa Cruz Biotechnology, Axil Scientific, Singapore) and phospho-P53 (Ser46) (GeneTex, Axil Scientific, Singapore). Chemiluminescence was imaged on ChemiDoc Touch Imaging System and analyzed with Image Lab Software V5.2.1 (Bio-Rad Laboratories, Singapore).
Sample preparation for TSP1 was modified: (1) 100 mM DTT used in sample loading buffer to break TSP1 homotrimer into monomer of 180 kDa; (2) methanol-free transfer buffer with 0.01% sodium dodecyl sulfate; (3) HSP90 was used as a high MW loading control. Culture supernatants were concentrated 40-fold using Amicon Ultra Centrifugal filters with a 50 kDa cutoff (Millipore, MERCK, Singapore) and loaded by equal volumes.
Zhou Y., Ng D.Y., Richards A.M, & Wang P. (2020). microRNA-221 Inhibits Latent TGF-β1 Activation through Targeting Thrombospondin-1 to Attenuate Kidney Failure-Induced Cardiac Fibrosis. Molecular Therapy. Nucleic Acids, 22, 803-814.
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2

Western Blot Analysis of TGF-β Signaling

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Cells were lysed directly in 4 × SDS sample buffer at 60–80% confluence. Lysates were subjected to standard SDS–PAGE. Bands were detected using enhanced chemiluminescence solution (Amersham). Secondary antibodies used throughout were horseradish peroxidase-conjugated polyclonal goat anti-mouse Ig (Dako, P0448, 1:2,000) and horseradish peroxidase-conjugated polyclonal goat anti-rabbit Ig (Dako, P0260, 1:2,000). Primary antibodies were PO4-SMAD3 (Abcam, 52903, 1:1,000), SMAD3 (Cell Signaling, 9523, 1:1,000), TGFBR1 (Santa Cruz, 398 (V22), 1:500) and TGFBR2 (Santa Cruz, 17792, (E6), 1:500). For TGFBR2 western blottings, lysates were prepared directly from transfected cells using the Dual-luciferase cell lysis buffer (Promega, UK). For TGFBR1 western blottings, parallel transfections to the luciferase assays were performed and samples were lysed directly in 4 × SDS sample buffer. Original uncropped western blot scans are also provided (Supplementary Fig. 15).
Cammareri P., Rose A.M., Vincent D.F., Wang J., Nagano A., Libertini S., Ridgway R.A., Athineos D., Coates P.J., McHugh A., Pourreyron C., Dayal J.H., Larsson J., Weidlich S., Spender L.C., Sapkota G.P., Purdie K.J., Proby C.M., Harwood C.A., Leigh I.M., Clevers H., Barker N., Karlsson S., Pritchard C., Marais R., Chelala C., South A.P., Sansom O.J, & Inman G.J. (2016). Inactivation of TGFβ receptors in stem cells drives cutaneous squamous cell carcinoma. Nature Communications, 7, 12493.
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3

Western Blot Analysis of EMT Markers

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For western blot analysis cells were lysed using RIPA buffer containing PhosSTOP Phosphatase Inhibitor Cocktail (Roche). Protein concentration was determined by Bradford assay (Bio‐Rad). Protein samples were separated by SDS-PAGE and transferred to nitrocellulose membranes (Amersham Biosciences). The following antibodies were used: E2F1 (KH‐95; 1:500), EGFR (1:250), FGFR1 (Flg C15; 1:1000), TGFBR1 (V22; 1:1000), TGFBR2 (L21; 1:1000), Vimentin (V9; 1:1500), ZEB1 (H‐102; 1:1000), Snail (H-130; 1:500), SMAD2/3 (FL-425; 1:1000), and c-SRC (1:500) from Santa Cruz, E‐Cadherin (1:1500), and NFKB1 (C22B4; 1:1500) from Cell Signaling; N-Cadherin (610921; 1:1500) and FN1 (1:1000) from BD Bioscience, Actin (Sigma; 1:4000) and their corresponding HRP‐conjugated secondary antibodies (Pierce; 1:2000). Detection of HRP activity was performed with the ChemiDoc TouchTM Imaging System (BioRad) using ECL Plus (Amersham) or Super Signal West Femto (Thermo Scientific) Western Blotting Detection Reagents (GE Healthcare). Uncropped pictures of the immunoblots are shown in Supplementary Figs 9, 10, and 11.
Khan F.M., Marquardt S., Gupta S.K., Knoll S., Schmitz U., Spitschak A., Engelmann D., Vera J., Wolkenhauer O, & Pützer B.M. (2017). Unraveling a tumor type-specific regulatory core underlying E2F1-mediated epithelial-mesenchymal transition to predict receptor protein signatures. Nature Communications, 8, 198.
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4

Western Blot Analysis of TGF-β Signaling

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Cell lysates were analysed by SDS-PAGE using the following antibodies: PO4-SMAD2 (Ser465/467) (rabbit polyclonal, #3101, Cell Signalling Technology [CST]), SMAD2 (mouse monoclonal, C16D3, CST), SMAD2/3 (mouse monoclonal, Clone 18, BD transduction Laboratories), SMAD4 (mouse monoclonal, B-8, Santa Cruz Biotechnology), TGFBR1 (rabbit polyclonal, V-22, Santa Cruz Biotechnology), CDKN1A (rabbit polyclonal, C19, Santa Cruz Biotechnology), RHOA (mouse monoclonal, 26C4, Santa Cruz Biotechnology), PO4-SRC (Tyr416) (rabbit monoclonal, D49G4, CST), SRC (rabbit monoclonal, 36D10, CST), PO4-p44/p42 MAPK (ERK1/2) (Thr202/Tyr404) (rabbit polyclonal, #9101, CST), p44/p42 MAPK (ERK1/2) (rabbit polyclonal, #9102, CST), β-actin (mouse monoclonal, AC-74, Sigma). Secondary HRP-conjugated antibodies (Dako) and enhanced chemiluminescence (GE Healthcare) was used to detect bound antibody.
Spender L.C., Ferguson G.J., Liu S., Cui C., Girotti M.R., Sibbet G., Higgs E.B., Shuttleworth M.K., Hamilton T., Lorigan P., Weller M., Vincent D.F., Sansom O.J., Frame M., Dijke P.T., Marais R, & Inman G.J. (2016). Mutational activation of BRAF confers sensitivity to transforming growth factor beta inhibitors in human cancer cells. Oncotarget, 7(50), 81995-82012.
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5

Western Blot Analysis of Gastric Cancer Markers

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Antibodies against fibronectin, DCX, E2F3, TGFBR1 and WEE1were purchased from Santa Cruz Biotechnology (CA, USA). phospho-Akt, SIRT1, FBXW7, SNAIL and vimentin were purchased from Abcam (Cambridge, UK), and total Akt, N-cadherin, E-cadherin, NTRK3, RELN, EGFR, GAPDH and BMI1were from BD Biosciences (USA). HRP-conjugated goat anti-rabbit IgG was purchased from Santa Cruz Biotechnology. Total protein was extracted from the transfected cells and gastric cancer tissues using RIPA lysis buffer (Beyotime, China) according to the manufacturer's instructions. After the whole-cell protein extracts were quantified using a BCA protein assay, equivalent amounts of cell lysates were resolved by 10% SDS polyacrylamide gel electrophoresis and were transferred onto a polyvinylidene fluoride membrane, which was then blocked in 5% non-fat milk in TBST for 1 h at 4°C. The blots were then incubated with primary antibodies. After incubation with HRP-conjugated secondary antibodies, the protein bands were visualized using an enhanced chemiluminescence reagent (Millipore, Billerica, MA, USA).
Yu W.W., Jiang H., Zhang C.T, & Peng Y. (2017). The SNAIL/miR-128 axis regulated growth, invasion, metastasis, and epithelial-to-mesenchymal transition of gastric cancer. Oncotarget, 8(24), 39280-39295.
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6

Immunohistochemical Analysis of TGF-β Signaling

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IHC was performed on formalin-fixed skin sections. Standard IHC techniques were used throughout this study. Primary antibodies were as follows: TGFBR1 (Santa Cruz, V22, 1:100), PO4-SMAD3 (Abcam, EP823Y, (52903), 1:50), GFP (Abgent, 168AT1211, 1:100), Keratin 1 (Covance, AF109, 1:1,000), Keratin 5 (Covance, AF138, 1:4,000), Keratin 15 (Abcam, 80522 (LHK15), 1:1,000), KI67 (Thermo, RM-9106-S) and BrdU (BD Biosciences, 347580, 1:200). Mouse PO4-SMAD3 score was performed in a blinded manner. For each antibody, staining was performed on at least three mice of each genotype and at least six sections of normal human skin. Representative images are shown for each staining. PO4-SMAD3 antibody was optimized for IHC use using formalin-fixed paraffin-embedded SCCIC4 cells treated with and without recombinant TGFβ1 or the TGFBR1 kinase inhibitor SB-431542 (ref. 70 (link)) (Supplementary Fig. 5). PO4-SMAD3 IHC scoring was performed in a blinded manner using the histoscore method.
Cammareri P., Rose A.M., Vincent D.F., Wang J., Nagano A., Libertini S., Ridgway R.A., Athineos D., Coates P.J., McHugh A., Pourreyron C., Dayal J.H., Larsson J., Weidlich S., Spender L.C., Sapkota G.P., Purdie K.J., Proby C.M., Harwood C.A., Leigh I.M., Clevers H., Barker N., Karlsson S., Pritchard C., Marais R., Chelala C., South A.P., Sansom O.J, & Inman G.J. (2016). Inactivation of TGFβ receptors in stem cells drives cutaneous squamous cell carcinoma. Nature Communications, 7, 12493.
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7

Western Blot Analysis of CEA, TGFβ Signaling

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Cells were lysed with lysis buffer and were standardized using a BCA protein assay (Pierce™ BCA Protein Assay Kit 23225). Equal amounts of proteins were fractionated on SDS–PAGE and blotted to nitrocellulose membrane. Membranes were incubated with CEA (Thermo MS-613-P0), TGFBR1 (Santa Cruz Sc-398), TGFBR2 (ab17650), Phospho-Smad3 (Ser423/425) (Cell Signaling #9520), Smad3 (Cell Signaling #9523), and tubulin (Cell Signaling #2144) antibodies. Secondary antibodies conjugated with horseradish peroxidase (Chemicon) and Enhanced chemiluminescence (ECL) kit (Perkin-Elmer Life Sciences) were used to develop the immunoblots.
Chen J., Raju G.S., Jogunoori W., Menon V., Majumdar A., Chen J.S., Gi Y.J., Jeong Y.S., Phan L., Belkin M., Gu S., Kundra S., Mistry N.A., Zhang J., Su X., Li S., Lin S.H., Javle M., McMurray J.S., Rahlfs T.F., Mishra B., White J., Rashid A., Beauchemin N., Weston B.R., Shafi M.A., Stroehlein J.R., Davila M., Akbani R., Weinstein J.N., Wu X, & Mishra L. (2016). Mutational Profiles Reveal an Aberrant TGF-β-CEA Regulated Pathway in Colon Adenomas. PLoS ONE, 11(4), e0153933.
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8

Investigating EMT Signaling Pathways

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Antibodies specific for TFAP2C, TGFBR1, tubulin, E-cadherin, vimentin, fibronectin, Snail, p-Snail, Smad3 and p-Samd3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies specific for PAK1, p-PAK1, MEK, p-MEK1, ERK and p-ERK were obtained from Cell Signaling Technology (Beverly, MA, USA). Cell culture media (RPMI-1640 and Dulbecco's modified Eagle's medium), fetal bovine serum (FBS), glutamine, penicillin and streptomycin were acquired from Gibco (Grand Island, NY, USA). Control small interfering RNA (siRNA) and siRNAs specific for TFAP2C #1 and TGFBR1 (ON-TARGETplus SMARTpool) were obtained from Dharmacon (Chicago, IL, USA) and siRNA specific for TFAP2C #2 (AccuTarget siRNA) was obtained from Bioneer (Daejeon, Republic of Korea).
Kim W., Kim E., Lee S., Kim D., Chun J., Park K.H., Youn H, & Youn B. (2016). TFAP2C-mediated upregulation of TGFBR1 promotes lung tumorigenesis and epithelial–mesenchymal transition. Experimental & Molecular Medicine, 48(11), e273-.
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9

Comprehensive Protein Expression Analysis

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Cell lysates were prepared with RIPA buffer (Cell Signaling, #9806), supplemented with 1 mM PMSF, 1 mM NaF, protease inhibitor cocktail (Roche, #04 693 132 001), and phosphatase inhibitor cocktail (Roche, #04 906 845 001). SDS–PAGE gels were transferred onto nitrocellulose membranes.
We used the following primary antibodies against: Phospho-SMAD2 (Ser465/467) (Cell Signaling, #3108), SMAD2 (Cell Signaling, #3103), β-Actin (Cell Signaling, #3700), TGFBR1 (Santa Cruz, #sc-398), TGFBR2 (abcam, #ab184948), and integrin β1 (Cell Signaling, #9699). All Western blot membranes were incubated with primary antibodies overnight at 4 °C. Secondary antibodies used for Western blotting were anti-rabbit IgG (H+L) DyLight 800 (Cell Signaling, #5151) and anti-mouse IgG (H+L) DyLight 680 (Cell Signaling, #5470). Western blot images were acquired and quantified using Odyssey CLx Imaging System (LI-COR Biosciences, #9140).
Li Y., Deng D., Höfer C.T., Kim J., Do Heo W., Xu Q., Liu X, & Zi Z. (2024). Liebig’s law of the minimum in the TGF-β/SMAD pathway. PLOS Computational Biology, 20(5), e1012072.
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10

Multimarker Immunofluorescence Analysis

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BMPR1A (1:20, sc20736; Santa Cruz); BRACHYURY/T (1:400, AF2085; R&D); Clover (1:600, EMU101; Kerafast); OCT4 (1:800, sc8628; Santa Cruz); pSMAD1/5 (1:800, 13820s; Cell Signaling); TGFBR1 (1:20, sc9048; Santa Cruz); ZO-1 (1:100, 33-9100; Thermo Fisher); ZO-1-FITC (1:100, 33-9111; Thermo Fisher).
Zhang Z., Zwick S., Loew E., Grimley J.S, & Ramanathan S. (2019). Mouse embryo geometry drives formation of robust signaling gradients through receptor localization. Nature Communications, 10, 4516.
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