P smad 2

The protein samples (10 μL) were separated by 10% SDS polyacrylamide gel electrophoresis, and transferred to a PVDF membrane (Millipore, Billerica, USA). Next, the membranes were blocked with 5% skim milk or 5% bovine serum albumin at 25°C for 2 h and then incubated with primary antibodies in the refrigerator overnight in a shaker at 4°C. The following antibodies were used: Kir2.1 (1:1000; Abcam), α-SMA (1:1000; Boster), collagen I (1:1000; Boster), collagen III (1:1000; Boster), TGF-β1 (1:1000; Abcam), Smad 2 (1:1000; Boster), p-Smad 2 (1:1000; Boster), Smad 3 (1:1000; Boster), p-Smad 3 (1:1000; Boster), and GAPDH (1:1000; Zhong Shan-Golden Bridge, Beijing, China). Next, the membranes were incubated with the corresponding HRP-conjugated secondary antibody at room temperature for 2 h, followed by three times wash with TBST for 10 min each. Finally, the ECL luminescence reagent (Biosharp, Hefei, China) was used to visualize the protein bands, and band density was analyzed on a Tanon-5200 gel imaging system (Tanon, Shanghai, China).

Protein lysates were isolated using lysis buffer (Beyotime Biotechnology, Shanghai, China) and quantified by the bicinchoninic acid method (Thermo Fisher Scientific, Waltham, MA, USA). Protein lysates (20 μg) were fractionated onto 10% SDS-PAGE (Sangon) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Whatman, Germany). PVDF membranes were blocked with 5% skim milk-TBS at 4°C overnight, incubated with specific primary antibody solutions at room temperature for 1 hour, and incubated with HRP goat anti-rabbit/ mouse IgG (1:20,000) at room temperature for 40 minutes. Enhanced chemiluminescence system (Millipore, Billerica, MA, USA) and Image-Pro Plus 6.0 software analysis system (Media Cybernetics, Inc., Bethesda, MD, USA) were used for protein blot quantitation. Antibodies against E-cadherin (1:1,000), N-cadherin (1:400), MDM2 (1:1,000), vimentin (1:1,000), β-catenin (1:5,000), Snail (1:500), Slug (1:1,000), Smad2/3 (1:2,000), p-Smad2 (1:1,000), p-Smad3 (1:2,000), and secondary antibodies were purchased from Boster Biotechnology (Wuhan, China). GAPDH antibody (1:10,000) was purchased from Yasunari Biological Engineering (Shanghai, China).