Gastric cancer cells with indicated treatment were rinsed with cold PBS before treated with RIPA lysis buffer (50 mM Tris, 0.15 M NaCl, 1 mM EGTA, 1% NP40, 0.25% SDS, pH 7.4) containing protease and phosphatase inhibitors. Cell pellets were incubated for 30 min on ice to homogenize fully and then total proteins in the supernatant of cell lysates were collected by centrifuging at 4°C at 12,000 rpm for 10 min.
Total protein concentrations were measured by the BCA protein assay kit (Pierce, USA) before experiment. Then, standard western blotting assays (SDS-PAGE) were used to analyze protein expression. The antibodies used for western blotting analysis in this study included: anti-PPP2R5A (Abcam, ab89621, 1:2,000 dilution), anti-c-Myc antibodies (Cell Signaling Technology, #5605, 1:1,000 dilution), anti-p-c-Myc (S62) antibodies (Cell Signaling Technology, #13748,1:1,000 dilution), anti-β-actin antibody (Sigma, A2228, 1:5,000 dilution). After incubation with corresponding species-speci c secondary HRP-conjugated antibodies, the target protein bands were visualized by an ECL imaging system.
Total protein concentrations were measured by the BCA protein assay kit (Pierce, USA) before experiment. Then, standard western blotting assays (SDS-PAGE) were used to analyze protein expression. The antibodies used for western blotting analysis in this study included: anti-PPP2R5A (Abcam, ab89621, 1:2,000 dilution), anti-c-Myc antibodies (Cell Signaling Technology, #5605, 1:1,000 dilution), anti-p-c-Myc (S62) antibodies (Cell Signaling Technology, #13748,1:1,000 dilution), anti-β-actin antibody (Sigma, A2228, 1:5,000 dilution). After incubation with corresponding species-speci c secondary HRP-conjugated antibodies, the target protein bands were visualized by an ECL imaging system.