Western Blot Analysis of ROR2 and Akt

Subconfluent cells were washed twice with PBS, and then lysed with ice-cold RIPA lysis buffer (50 mmol/L Tris, 150 mmol/L NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mmol/L sodium orthovanadate, 1 mmol/L sodium fluoride, 1 mmol/L EDTA, 1 mmol/L PMSF, and 1% cocktail of protease inhibitors) (pH7.4). The lysates were then clarified by centrifugating at 12,000g for 20 min at 4 °C. The protein extracts were separated by SDS-PAGE. The immunoblotting procedure was performed as described [16 (link)] and the following antibodies were used: rabbit anti-ROR2 antibody, mouse anti-GAPDH antibody (Proteintech, Wuhan, China), rabbit anti-Akt antibody, rabbit anti-phospho-Akt (p-Ser473) antibody (Cell Signaling Technology, Danvers, MA). Protein bands were detected by incubating with horseradish peroxidase-conjugated antibodies (Santa Cruz Biotechnology) and visualized with ECL reagent (Thermo Scientific, Rockford, IL). The gray values were taken by Tanon imaging analysis system (Tanon, Shanghai, China).