Lysates

Expression of Bcl-2, Bax, caspase-3, MAPK (p38, p-p38, ERK and p-ERK) and phosphoinositide 3-kinase (PI3K)/Akt/mTOR signaling protein (Akt, p-Akt, mTOR and p-mTOR) levels in MGC-803/maspin⁻ cells was examined by western blot analysis. Cells were centrifuged (8000 × g, 15 min, 4°C), washed with cold PBS and lysed on ice for 30 min in lysates (Beyotime Institute of Biotechnology) containing 100 µg/ml phenylmethanesulfonyl fluoride. Protein concentrations were determined by NanoPhotometer pearl (P-330-31; Implen GmbH). Total protein (20–70 µg) was electrophoresed on 8–12% SDS-PAGE and then transferred to a nitrocellulose membrane. Following incubation with 5% non-fat dried milk for 2 h at room temperature, Primary antibodies were diluted with 5% skimmed milk powder solution and incubated with the membranes overnight at 4°C, then washed three times with Tris-buffered saline (pH 6.8) with Tween-20 (0.1%) (TBST), incubated with the secondary antibody (sheep anti-rabbit IgG-HRP; cat no. HA1001; 1:2,000; Hangzhou HuaAn Biotechnology Co., Ltd. Hangzhou, China) at room temperature for 2 h and washed three times with TBST. The ECL reagent was used to visualize the positive bands on the membrane.