Anti tgf r1 antibody

Stable cell lines (10 7 cells) were harvested and suspended in immunoprecipitation buffer [50 mM HEPES (pH 7.6), 250 mM NaCl, 5 mM EDTA (pH 8.0), 0.1% NP-40, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail] and sonicated. After centrifugation at 13,000 rpm for 15 min at 4 °C, the supernatant was transferred into a new tube and precleared with 50 L of protein G-agarose beads (Roche). Next, the precleared supernatant was incubated with 50 L of protein G-agarose beads with antibodies against NSrp70 (ALTAS) or IgG (Sigma), overnight at 4 °C, and washed with immunoprecipitation buffer. For the ubiquitin experiment in vitro, anti-TGF R1 antibody was used (Abcam). To harvest the protein complex, 50 L of 1 SDS loading buffer (62.4 mM Tris, pH 6.8, 10% glycerol, 2% SDS, and 0.0012% bromophenol blue) was added, the mixture incubated for 10 min at 95 °C, and analyzed by western blotting.

MDA-MB231 and PC-3 tumor cell lines and COS-7 immortalized cells were obtained from the American Type Culture Collection and cultured in a humidified incubator with 5% CO 2 at 37°C in Dulbecco's modified Eagle's medium or RPMI 1640 media supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin, respectively. Antibodies directed against Sema4C (sc-136445), N-cadherin (sc-7939), fibronectin (sc-8422), Zeb1 (sc-25388), SMAD1 (sc-7965), ID1 (sc-488), actin (sc-1616), PlexinB2 (sc-34504), TGF--R2 (sc-17792), and ACTR1/ACVR1 (sc-374523) were all from Santa Cruz Biotechnology. Anti-vimentin (VIM-3B4) was from Merck. Anti-p-SMAD1/5/9 (D5B10, no. 13820) and anti-p-SMAD2/3 (D27F4, no. 8828) were from Cell Signaling Technology. Anti-vinculin (V4505) and anti-Flag (clone M2_F3165) were from Sigma-Aldrich. Anti-E-cadherin was from BD Transduction Laboratories (catalog no. 610182). Anti-TGF--R1 antibody was from Abcam (ab31013). Anti-BMPR1A was purchased from Proteintech (catalog no. 12702-1-AP). Cell treatments with 1 to 5 M SB-431542 hydrate (Sigma-Aldrich) were performed for 48 to 72 hours, as described. Purified recombinant Sema4C ectodomain (catalog no. AF6125) was purchased from R&D Systems. A tumor multiple tissue array containing primary and metastatic breast cancer samples (BR1008A) was obtained from US Biomax Inc. (Rockville, MD).