Anti tgfβr1

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-TGFβR1 is a primary antibody that recognizes the transforming growth factor beta receptor 1 (TGFβR1) protein. TGFβR1 is a serine/threonine protein kinase that plays a crucial role in the TGF-beta signaling pathway.

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4 protocols using anti tgfβr1

Cartilage Protein Extraction and Analysis

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The protein was extracted from 10 mg articular cartilage using a protein isolation kit (ReadyPrep; GE Healthcare Life Sciences). The protein concentration was determined using a bicinchoninic assay kit (Thermo Fisher Scientific, Inc.). The protein (20 μg) was separated on 12% SDS PAGE gel and transferred to the nitrocellulose membrane. At room temperature, the membrane was sealed in 5% skimmed milk for 2 hours and incubated with the following primary antibodies at 4°C overnight: Anti-LC3 I, Anti-LC3 II, anti-MMP13, anti-aggrecan, anti-collagen II, anti-TGFβR1, anti-β-actin (Cell Signaling Technology, Inc., Danvers, USA). The next day, the nitrocellulose membranes were washed three times and incubated with HRP-labeled goat anti-rabbit IgG secondary antibody (1:10,000, cat. no. A16104SAMPLE; Thermo Fisher Scientific, Inc.) at 4°C for 2 h. The protein bands were detected by an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.) scanned by ChemiDoc XRS (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein expression was normalized to β-actin and densitometric analysis was performed by ImageJ Software version 7.0 (National Institutes of Health, Bethesda, MD, USA).

Tao X.M., Liu P.F., Gu H.Y., Lian D.B., Gao L., Tao W.W., Yan D, & Zhao B. (2020). Cordycepin Alleviates Anterior Cruciate Ligament Transection (ACLT)-Induced Knee Osteoarthritis Through Regulating TGF-β Activity and Autophagy. Drug Design, Development and Therapy, 14, 2809-2817.

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Western Blot Analysis of TGFβ Signaling

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Cells were harvested in lysis solution, as previously described [46 (link)], and subjected to Western blotting analysis. Membranes were probed with the following primary antibodies: anti-phosphorylated smad2, anti-total smad2, anti-TGFβR1, anti-phosphorylated Chk2, anti-total Chk2, anti-PARP, anti-CD44, anti-Olig2 (all from Cell Signaling, Boston, MA, USA), and anti-APOBEC3G (Proteintech, Chicago, IL, USA). Anti-β-actin antibody was purchased from Sigma (St. Louis, MO, USA) and used as the loading control.

Wang Y., Wu S., Zheng S., Wang S., Wali A., Ezhilarasan R., Sulman E.P., Koul D, & Alfred Yung W.K. (2017). APOBEC3G acts as a therapeutic target in mesenchymal gliomas by sensitizing cells to radiation-induced cell death. Oncotarget, 8(33), 54285-54296.

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Immunoprecipitation of Signaling Proteins

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Cells were lysed in cell lysis buffer (50 mM Tris-HCL pH7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 100 mM KCl, 50 mM NaF, 10 mM Na 2-glycerophosphate, 1 mM Na₃VO₄, and supplemented with protease inhibitor set (Roche)). For immunoprecipitation, 1 mg cell lysate was used to incubate with 1µg/ml of antibodies. Immunocomplex was precipitated using protein A/G sepharose (Thermo Fisher Scientific, Waltham, MA). Antibodies used are as follows: anti-p-SMAD2/3, anti-p-JNK1/2, and anti-TGFβR1 (Cell Signaling Technology); anti-SMAD1/2/3, anti-JNK1/2, and anti-RYK antibodies (Santa Cruz Biotechnologies); anti-WNT5A (Genetex); and anti-α-tubulin (Sigma Aldrich); anti-keratin 8 (clone TROMA-I, Developmental Studies Hybridoma Bank at the University of Iowa).

Borcherding N., Kusner D., Kolb R., Xie Q., Li W., Yuan F., Velez G., Askeland R., Weigel R.J, & Zhang W. (2015). Paracrine WNT5A signaling inhibits expansion of tumor-initiating cells. Cancer research, 75(10), 1972-1982.

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Western Blot Analysis of EMT Markers

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The cells were harvested and washed with PBS buffer, then lysed by 1 × SDS loading buffer. The lysates were boiled at 100 °C for 5 min. The samples were centrifuged at 10,000 rpm for 1 min. Around 50 μg of total proteins was loaded onto SDS-PAGE gel and resolved. After that, the proteins were transferred to PVDF membrane at 300 mA for 1.5 h. The membrane was blocked with 5% non-fat milk in 1× TBST for 1 h at room temperature, the membrane was then incubated with primary antibodies at 4 °C overnight. The following day, the membrane was washed with 1× TBST for three times, 5 min each time. The membrane was incubated with secondary antibodies at room temperature for 1 h. Finally, the membrane was incubated with ECL solution and then exposed. The following antibodies were used: anti-TGFβR1 (cell signaling technology, USA), anti-E-cadherin (cell signaling technology, USA), anti-N-cadherin (cell signaling technology, USA), anti-Claudin (cell signaling technology, USA), anti-β-actin (Proteintech, USA), anti-MMP9 (Abcam, USA), anti-GAPDH (Proteintech, USA).

Mu Y., Li N, & Cui Y.L. (2018). The lncRNA CCAT1 upregulates TGFβR1 via sponging miR-490-3p to promote TGFβ1-induced EMT of ovarian cancer cells. Cancer Cell International, 18, 145.

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