Western blot detection of apoptotic proteins

GCSCs transfected with miRNAs or treated with drugs were harvested, lysed with lysis buffer (1 % Triton X-100, 1 mM EGTA, 0.5 % NP-40, 10 mM HEPES (pH 7.4), 1 mM EDTA, 0.15 M NaCl) supplemented with protease inhibitor cocktail (Cell Signaling Technology, Beverly, MA, USA), and sonicated on ice for 10 s. After incubation on ice for 30 min, the lysate was centrifuged at 13,000 rpm for 15 min at 4 °C, the supernatant was collected, and the protein concentration was determined using the Bicinchoninic Acid (BCA) Protein Assay Kit (Pierce, Rockford, IL, USA). Then, 50 g of each lysate was boiled in 5× loading buffer (50 % glycerol, 1 M Tris–HCl, pH 6.8, 10 % β-mercaptoethanol, 10 % SDS, 1 % bromophenol blue), cooled on ice, and separated by SDS-PAGE in 12 % gels. The resolved proteins were analyzed by western blotting using anti-BIM and anti-BAK antibodies (Abcam, Cambridge, MA, USA) and their corresponding secondary antibodies.