Cells were seeded at 500,000 cells per 10-cm dish and treated the next day. After treatment for 6 and 16 hours, cells were scraped off the wells in cold PBS, centrifuged at 5000 rpm for 3 min at 4°C, separated from the supernatant, washed with PBS once, frozen at −80°C, and then lysed with cold radioimmunoprecipitation assay buffer [20 mM tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 1% NP-40, 0.1% SDS, 0.5% nadeoxycholate, 2 mM EDTA (pH 8.0)] supplemented with protease and phosphatase inhibitors (Roche protease inhibitor cocktail; Phosphatase Inhibitor Cocktail I and Phosphatase Inhibitor Cocktail II from Sigma-Aldrich) and centrifuged at 13,300 rpm at 4°C for 10 min. Protein concentration in supernatant lysates was determined using the Bradford assay (Bio-Rad). Proteins from each lysate (10 μg) were resolved on SDS-PAGE (NuPAGE 4 to 12%), transferred to polyvinylidene difluoride membranes, blocked with 5% milk in tris-buffered saline + 0.1% Tween, and probed with primary antibodies in 5% bovine serum albumin overnight at 4°C. Primary antibodies (1:1000 to 1:2000 dilution) recognized BCL-XL (CST#2764); BIM (CST#2933); BID (CST# 2002); caspases 3, 8, and 9; and β-actin.

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