Western Blot Analysis of RA-FLS Proteins

The western blot procedure was carried out as previously described [25 (link)]. Briefly, RA-FLS were washed twice with cold PBS and lysed in RIPA lysis buffer containing 50 mM Tris, 0.15 M NaCl, 1 mM EGTA, 1% NP40, 0.25% SDS (Beyotime Institute of Biotechnology, Haimen, China), protease inhibitor mix, and phosphatase inhibitors (Roche Diagnostics, Switzerland). Samples were boiled and resolved with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, USA). Membranes were immunoblotted with primary antibodies followed by incubation with the horseradish peroxidase- (HRP-) conjugated secondary antibodies. The protein bands were visualized with the Pierce ECL Plus western blotting substrate (Thermo Fisher Scientific, Inc., USA), and GAPDH was used as the loading control. The following antibodies were used in the present study: rabbit anti-YAP (no. 14074), rabbit anti-TAZ (no. 4883), rabbit anti-ULK1 (no. 8054), rabbit anti-LC3B (no. 3868), rabbit anti-SQSTM1/p62 (no. 39749), rabbit anti-β-catenin (no. 8480), rabbit anti-Vimentin (no. 5741), HRP- linked anti-rabbit IgG (no. 7074), anti-mouse IgG (no. 7076) purchased from Cell Signaling Technology (Danvers, MA, USA), and mouse anti-GAPDH purchased from KangChen Bio-tech (Shanghai, China).