Tgfbr2

Manufactured by Cell Signaling Technology

Sourced in United States

TGFBR2 is a receptor protein that binds to the transforming growth factor-beta (TGF-β) ligand. It is a key component of the TGF-β signaling pathway, which regulates various cellular processes, including cell growth, differentiation, and apoptosis.

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Anti-TGFBR2 (4 citations)

14 protocols using tgfbr2

Protein Extraction and Antibody Analysis

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Protein was extracted according to the previous method.⁵⁷ (link) Primary antibodies against Col1a1, α-SMA, VIM, Hsp-90, GAPDH (all from Cell Signaling Technology, MA), Smad2, p-Smad2, Smad3, p-Smad3, Tgfbr2 (all from Cell Signaling Technology, MA), and anti-tubulin (Sigma) antibodies were used as primary antibodies.

Sun Y., Wang H., Li Y., Liu S., Chen J, & Ying H. (2018). miR-24 and miR-122 Negatively Regulate the Transforming Growth Factor-β/Smad Signaling Pathway in Skeletal Muscle Fibrosis. Molecular Therapy. Nucleic Acids, 11, 528-537.

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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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Protein Extraction and Immunoblotting

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Cells were lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton-X 100, 1 mM each MgCl₂, MnCl₂ and CaCl₂, 1 mM PMSF and 10 mM sodium fluoride) [23 (link),24 (link)]. Tissues were homogenized in T-PER tissue protein extraction reagent (Thermo, 78510), in which protease inhibitor cocktail (Biotool, B14001) was added. Insoluble material was removed by centrifugation at 10,000 g for 10 min. Proteins were separated by SDS-PAGE under reducing condition, followed by blocking in phosphate-buffered saline/Tween-20 containing 1% BSA. The membrane was incubated with antibodies for CTHRC1 (Huabio), TGFBR1 (Cell Signaling, 3712S), TGFBR2 (Cell Signaling, 11888S), TGFBR3 (Cell Signaling, 2519S), Endoglin (Cell Signaling, 4335S), phospho-Smad2 (Cell Signaling, 3108P), Smad2 (Cell Signaling, 5339P), phospho-Smad3 (Cell Signaling, 9520P), Smad3 (Cell Signaling, 9523P), Smad4 (Cell Signaling, 9515P), phospho-TAK1 (Cell Signaling, 4508S), TAK1 (Cell Signaling, 5206S), phospho-p38 (Cell Signaling, 4511S), p38 (Cell Signaling, 8690S), phosphor-JNK (Cell Signaling, 4668S), JNK (Cell Signaling, 9252S), Wnt5a (Cell Signaling, 2392S), and GAPDH (Huabio, M1211-1), followed by incubation of species-specific secondary antibodies. Bound secondary antibodies (LI-COR, 926-32213; 926-68051) were revealed by Odyssey imaging system (LI-COR).

Li J., Wang Y., Ma M., Jiang S., Zhang X., Zhang Y., Yang X., Xu C., Tian G., Li Q., Wang Y., Zhu L., Nie H., Feng M., Xia Q., Gu J., Xu Q, & Zhang Z. (2019). Autocrine CTHRC1 activates hepatic stellate cells and promotes liver fibrosis by activating TGF-β signaling. EBioMedicine, 40, 43-55.

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Protein Extraction and Western Blotting

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Proteins from transfected cells were extracted using RIPA buffer (Sigma) supplemented with Protease Inhibitor Cocktail (Roche). The protein concentrations of the lysed samples were measured using the bicinchoninic acid assay (Bio‐Rad). The proteins were electrophoresed on a 10% SDS‐PAGE and were then transferred to the polyvinylidene difluoride (PVDF) membranes. After incubating with 5% skimmed milk for 1 hour at room temperature, the PVDF membranes were probed with primary antibodies against TGFBR2 (Cell Signaling Technology) and β‐actin (Cell Signaling Technology) by a further overnight incubation at 4°C. Afterwards, the PVDF membranes were washed with phosphate buffered saline with Tween‐20 for 3 times × 5 mins and were then incubated with horseradish peroxidase‐labelled secondary antibodies (Cell Signaling Technology). The Western blot signals were visualized using the ELC Substrates (ThermoFisher Scientific).

Xu H., Zhang B., Yang Y., Li Z., Zhao P., Wu W., Zhang H, & Mao J. (2020). LncRNA MIR4435‐2HG potentiates the proliferation and invasion of glioblastoma cells via modulating miR‐1224‐5p/TGFBR2 axis. Journal of Cellular and Molecular Medicine, 24(11), 6362-6372.

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Western Blot Analysis of Signaling Proteins

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Anti-Smad2 (#5339), p-Smad2 (Ser465/467 #3108), p-Akt (Thr308, #2965), p-Akt (Ser473, #4060), NFkB (#3037), p-Erk (Ser259 #9911), p-p38 (#4511), p-Jnk (#4668), p-Stat3 (#9145), p-Stat1 (#9171), Tgfbr2 (#3713), p-IRS1 (Tyr895, 3070), p-Smad1/5(#9511), p-Pten (Ser380/Thr382/383, #9549), Pten (D4.3 #9188), and Act-b (13E5, #4970) antibodies were purchased from Cell Signaling Technology. Anti-p-Akt (T308) antibody (# 658320) was purchased from R&D Systems. Anti-Mycn (B8.4.B, #sc-53993) antibody was purchased from Santa Cruz Biotechnology. Anti-pCKIIβ (S209) antibody (STJ90892) was purchased from St Joh’s laboratory. Anti-Gapdh antibody (10R-2932) was purchased from Fitzgerald Industries International. Western blot analysis was performed according to the standard procedure. Uncropped blots are included in Supplemetary Figs. 9–12.

Mirzamohammadi F., Kozlova A., Papaioannou G., Paltrinieri E., Ayturk U.M, & Kobayashi T. (2018). Distinct molecular pathways mediate Mycn and Myc-regulated miR-17-92 microRNA action in Feingold syndrome mouse models. Nature Communications, 9, 1352.

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Western Blot Analysis of TGF-β Signaling

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Cells were lysed using RIPA buffer composed of 50 mM Tris–HCl (pH 7.5), 0.25% Na‐deoxycholate, 1% Nonidet P‐40, 150 mM NaCl, 1 mM EDTA, and complete protease inhibitor cocktail (Roche, Indianapolis, IN). After centrifugation at 14,000 rpm for 10 minutes, total protein from the supernatant was collected and measured using the BCA protein assay kit (Pierce, Rockford, IL). The amount of 20 μg of protein sample was loaded on a 7.5% polyacrylamide gel (Bio‐Rad) for electrophoresis and then transferred onto a polyvinylidene fluoride membrane (Bio‐Rad). The membrane was incubated with blocking solution composed of 5% nonfat milk (Bio‐Rad) and 0.1% Tween 20 (Sigma–Aldrich) in Tris‐buffered saline overnight at 4°C with primary antibody against glyceraldehyde 3‐phosphate dehydrogenase (GAPDH), BMPR2, TGFBR2, SMAD3, pSMAD3, SMAD1, or pSMAD1/5 (Cell Signaling, Danvers, MA) before incubated with horseradish peroxidase‐linked secondary antibody (Cell Signaling) for 1 hour, detected by SuperSignal West Pico Chemiluminescent Substrate (Pierce), and imaged using the Kodak image station 4000R Pro (Kodak, Rochester, NY).

Wang J.F., Lee M., Tsai T., Leiferman E.M., Trask D.J., Squire M.W, & Li W. (2019). Bone Morphogenetic Protein‐6 Attenuates Type 1 Diabetes Mellitus‐Associated Bone Loss. Stem Cells Translational Medicine, 8(6), 522-534.

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Activin A and TGFβ1 Signaling Analysis

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Activin A was reconstituted in PBS; TGFβ1 in 4 mM HCl according to the manufacturer’s instruction (both R&D, Minneapolis, MN, USA). Final concentrations used were 25 ng/ml and 10 ng/ml, respectively, as previously described [31 (link), 49 (link)–51 (link)]. For inhibition of PI3K, we used LY294002 and for inhibition of MEK1/2, U0126 (both Cell Signaling Technology, Danvers, MA, USA). For immunoprecipitation and Western blotting, we used antibodies against ACVR1B (Santa Cruz Biotechnology, Santa Cruz, CA, USA), ACVR2A (customized by Yenzym, San Francisco, CA, USA), p85 (# 4292), TGFBR1 (# 3712) or TGFBR2 (# 3713, all Cell Signaling), p21 (# sc-65595, Santa Cruz), pan-Akt (# 8805, Abcam, Cambridge, MA, USA), GAPDH (# sc-47724, Santa Cruz), E-Cadherin (# 3195), vimentin (# 3390), pAkt Ser473 (# 4060) and pAkt Thr308 (# 13842, all Cell Signaling). For immunohistochemical analyses, we used p21 (# sc-817, Santa Cruz) ACVR2A (# ab10595), TGFBR2 (# ab78419), pAkt Ser473 (# ab81283, abcam), and pERK1/2 (# ab50011, all Abcam).

Bauer J., Ozden O., Akagi N., Carroll T., Principe D.R., Staudacher J.J., Spehlmann M.E., Eckmann L., Grippo P.J, & Jung B. (2015). Activin and TGFβ use diverging mitogenic signaling in advanced colon cancer. Molecular Cancer, 14, 182.

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ACVR1B and ACVR2A Immunoprecipitation

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1 mg of total protein lysate from various treatments were incubated with 1 μg of ACVR1B or ACVR2A antibody (Yenzym); ligand specificity was confirmed with TGFBR1 or TGFBR2 (Cell Signaling) overnight at 4 °C. Protein A beads (Invitrogen, Carlsbad, CA, USA) were added for 6 h. After denaturation with sample buffer, equivalent protein was fractionated on 4-20 % gradient gels (Biorad, Hercules, CA, USA), transferred to membranes and blotted with antibodies against ACVR1B antibody (Santa Cruz), and p85 (Cell Signaling).

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Immunoblotting Analysis of Cell Signaling

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Cell extracts were cleaned with 1× PBS buffer, prepared with RIPA lysis buffer, and analyzed by immunoblotting. Antibodies of cleaved caspase 3, cleaved PARP, BCL2, BAX, E-cadherin, ICAM-1, Vimentin, Twist 1, MMP2, MMP9, GAPDH, SMAD2, SMAD3, p-SMAD2, p-SMAD3, TGFBR1, and TGFBR2 were purchased from CST (Cell Signaling Technology, Danvers, MA, USA) and the secondary goat anti-rabbit antibody was obtained from Santa (Cruz Biotechnology, Dallas, TX, USA). LabWorks^TM Image Acquisition and Analysis Software (UVP, Upland, CA) were used to quantify band intensities.

Zhou B., Guo W., Sun C., Zhang B, & Zheng F. (2018). Linc00462 promotes pancreatic cancer invasiveness through the miR-665/TGFBR1-TGFBR2/SMAD2/3 pathway. Cell Death & Disease, 9(6), 706.

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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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