Anti tgfbr2

After treatment for the indicated time, porcine GCs were collected and western blotting assays were performed as we previously described [24] . In brief, the total protein from porcine GCs were extracted by ice-cold RIPA lysis buffer with 1% PMSF, and the concentration of total protein were measured by BCA method. Then, equal amount (~15 μg) of total protein was separated on an 4-20% SDS-PAGE gel and subsequently transferred into PVDF membrane (Merck Millipore, Germany) after electrophoresis. After incubation with 5% non-fat milk at room termperature for 90 min, the membranes were incubated with primary antibodies at 4°C overnight and then with corresponding secondary antibodies for 1 h. The images were obtained after incubation with ECL reagent. The primary antibodies used here were as follows: anti-SMAD4 (1:1000, #10231-1-AP, Proteintech, Jiangsu, China), anti-ACVR1B (1:1000, #D120045, Sangon Biotech, Shanghai, China), anti-BMPR2 (1:1000, #D221406, Sangon Biotech, Shanghai, China), anti-TGFBR2 (1:800, #sc-400, Santa Cruz, USA), anti-CREB1 (1:1000, #9197, Cell Signaling Technology, USA), anti-c-JUN (1:2000, #9165, Cell Signaling Technology, USA), anti-SP1 (1:1000, #2250, Cell Signaling Technology, USA) and anti-GAPDH (1:3000, #TA802519, ORIGENE, Jiangsu, China).