Tgf βrii

The protein lysates were separated on SDS-PAGE gels and then transferred to PVDF membranes (Invitrogen, Waltham, United States). The membranes were blocked in 5% BSA for 1 h and incubated with following primary antibodies: TNF-α (1:1,000, Santa Cruz, California, United States), P65, IκB, and p-IκB (1:1,000, Abmart, Shanghai, China), E-cadherin (1:1,000, Affinity, Nanjing, China), SMAD4 (1:1,000, Cell Signaling Technology, Danvers, United States), α-SMA (1:1,000, Merck, Darmstadt, Germany), FN (1:1,000, Abcam, Cambridge, United Kingdom), TGF-βRII, SMAD2/3, p-SMAD2/3 (1:1,000, Elabscience, Wuhan, China), and β-actin (1:1,000, Santa Cruz, California, United States) overnight at 4°C. The membranes were then washed three times with TBST for 10 min and incubated with secondary antibody (1:5,000, ZSGB-BIO, Beijing, China). The protein bands were visualized using ECL reagents.

MLg was washed three times with PBS and lysed using a cell lysis reagent. Proteins were analyzed by SDS-PAGE (10% polyacrylamide gels) and transferred to PVDF membranes (Invitrogen, United States). Membranes were blocked with 5% BSA for 1 h and incubated with primary antibodies, which were against α-SMA (Merck, GER; 1:1,000 dilution), FN (Abcam, United States; 1:1,000 dilution), TGF-βRII (Elabscience, CHN; 1:1,000 dilution), SMAD2/3 (Elabscience, CHN; 1:1,000 dilution), p-SMAD2/3 (Elabscience, CHN; 1:1,000 dilution), and β-actin (Santa Cruz, United States; 1:1,000 dilution). The membranes were then washed three times with TBST and incubated with goat anti-rabbit or anti-mouse secondary antibody (ZSGB-BIO, CHN; 1:5,000 dilution) at room temperature for 1 h. The bands of protein were obtained from the same membrane, which were then visualized using an ECL kit on a Tanon-5200 chemiluminescence detection system (Tanon, CHN) and quantified using ImageJ program.