Anti phosphorylated smad3

Total protein was extracted using RIPA plus buffer. The protein concentration was measured using BCA Protein Assay Kit (Takara, Dalian, China) as manufacturer’s instruction. 30 μg proteins were electrophoresed on 10% SDS-PAGE, and transferred to PVDF membrane (Sigma-Aldrich). The PVDF membranes were blocked with 5% non-fat milk at room temperature for 1 hr, and incubated with the primary antibodies at 4°C overnight. Following by incubation with goat anti-rabbit IgG antibody (1:10000 dilutions, Cell Signaling Technology) marked with horseradish peroxidase at room temperature for 2 hrs. Membranes were scanned by using an Odyssey Imaging System and analyzed with Odyssey v2.0 software (LICOR Biosciences, Lincoln, NE, USA). The primary antibodies used in this study are as follows: anti-phosphorylated p65 (1:1000 dilution, Cell Signaling Technology), anti-p65 (1:1000 dilution, Cell Signaling Technology), anti-Bcl-2 (1:1000 dilution, Cell Signaling Technology), anti-active-caspase3 (1:1000 dilution, Abcam Cambridge, MA, USA), anti-phosphorylated Smad2 (1:1000 dilution, Cell Signaling Technology), anti-phosphorylated Smad3 (1:1000 dilution, Cell Signaling Technology), anti-E-cadherin (1:1000 dilution, Cell Signaling Technology), anti-N-cadherin (1:1000 dilution, Cell Signaling Technology) and anti-β-actin (1:1000 dilution, Cell Signaling Technology).