Co-immunoprecipitation

For co-immunoprecipitation studies HEK293T cells were transfected with the indicated plasmids in 10-cm plates. 48 h post-transfection cells were treated with TGF-β (5–10 ng/mL) for 30–60 min. Cells were washed with ice cold PBS twice and lysates were made in 1 ml IP-lysis buffer supplemented with 1× PhosStop (Roche), 1× Protease inhibitor cocktail (Roche), Sodium Ortho Vanadate, Sodium fluoride, PMSF, and β-Glycerol phosphate (2 µM each). Protein estimation was performed using detergent compatible Dc assay kit (Pierce). 400–500 µg lysates were pre-cleared by incubating with normal Rabbit-IgG or normal Mouse-IgG for 1 h followed by 30 µl protein A/G-coupled Sepharose beads (GE Healthcare) for an additional hour at 4 °C and centrifuged. Co-immunoprecipitation was carried out by incubating precleared cell lysate (400 µg protein) with 1–2 µg Myc-tag, TGFBR1 or TGFBR2 specific antibodies overnight at 4 °C on a rotating platform. The immune complex was captured using 30 µl slurry of protein A/G-PLUS Sepharose beads for 2–4 h, washed four times with lysis buffer. Alternatively, FL-beads were used for 2 h at room temperature for pull-downs. After washing, the beads were boiled in 60–80 µl 1.2× Laemmli buffer diluted with IP-lysis buffer containing protease and phosphatase inhibitors to elute bound protein complexes. The Laemmli buffer was supplemented with 1–2% β-mercaptoethanol and 0.01% SDS to denature the proteins. The resulting pellet was resolved by 4–12% Bis-Tris SDS-PAGE gels in MOPS buffer and transferred to PVDF membrane for western-analysis.