Rabbit anti p smad2

For western blot assays, cells were harvested and sonicated in IP buffer (1% Triton X-100, 80 mM NaCl, 3 mM EDTA, and 50 mM HEPES with protease inhibitors). Generally, 5∼20 µg of protein from lysate supernatants was separated by size, blotted with each primary and secondary antibody, and detected with WEST-ZOL plus (Intron). Rabbit anti-β-actin (Sigma), rabbit anti-p-Smad2 (Cell Signaling, #3101), rabbit anti-Smad2 (Cell Signaling, #5339), and rabbit anti-p-Smad2 (Cell Signaling, #3101) were used for this research.

Cells from untreated group, TGF-β1-treated and SB431542-treated cells were washed 3 times with ice-cold PBS. Lysis buffer was then added, and the cells were lysed on ice for 30 min, followed by centrifugation at 12,000 rpm for 10 min at 4°C to remove cell debris. Protein samples were mixed with loading buffer and heated at 100°C for 10 min, after which the samples were separated by 10% SDS-PAGE gels (Bio-Rad, California, USA). Proteins were then transferred to polyvinyl difluoride membranes (Solarbio Systems, Beijing, China). Membranes were blocked in 5% nonfat milk in TBST buffer for 1–2 h at room temperature. The blots were incubated with primary antibodies overnight at 4°C with rabbit anti-p-Smad2 (1∶1000, Cell Signaling Technology, Massachusetts, USA), anti-Smad7 (1∶200, Santa Cruz Biotechnology, California, USA), anti-E-cadherin (1∶500, Abcam, Cambridge, United Kingdom), mouse anti-vimentin (1∶100, Santa Cruz Biotechnology, California USA), or anti-N-cadherin antibodies (1∶1000, Abcam, Cambridge, United Kingdom)This procedure was followed by incubation with sheep anti-mouse (1∶20000) or anti-rabbit (1∶20000) HRP-labeled secondary antibodies for 2 h at room temperature. The bands were detected with an ECL reagent kit (Thermo Systems, Massachusetts, USA). β-Actin immunoblots served as a loading control. All experiments were repeated three times.