Ripa buffer

Cells were harvested, washed in PBS, and re-suspended in RIPA buffer (Tebu-BIO #AR0105) in the presence of a protease inhibitor cocktail (Thermo Scientific™, Madrid, Spain #A32955). After incubation on ice for 30 min, the lysates were centrifuged at 14,000× g for 10 min at 4 °C and the supernatant fractions were used for Western blot analysis. Protein concentration was measured by Bradford reagent (Bio-Rad protein assay) using bovine serum albumin as a standard. Protein extracts (30 μg/lane) were resolved on a 10% SDS-polyacrylamide gel and electro-blotted onto PVDF membranes (AmershamTM ProtranTM 0.45 μm NC). Membranes were blocked in 5% low-fat milk in TBST or 5% BSA (50 mM Tris pH 7.5, 0.9% NaCl, 0.1% Tween 20) for 1 h at room temperature and probed overnight at 4 °C with a rabbit polyclonal anti-Bcl-2 (1:1000) (Cell Signaling, #2872), rabbit polyclonal anti-p70 S6 kinase (1:1000) (Cell Signaling, #9202), rabbit polyclonal anti-phospho-p70 S6 kinase (Ser371) (1:1000) (Cell Signaling, #9208), or mouse monoclonal anti-GAPDH (1:1000) (Santa Cruz, sc-47724). Horseradish peroxidase conjugated anti-mouse or anti-rabbit IgGs (1:5000 in blocking solution) (Santa Cruz, Spain) were used as secondary antibodies. Immunodetection was carried out using Bio-Rad chemiluminescent substrates and recorded using a Gel Documentation System (Syngene).

Total protein extracts were prepared from 500,000 cells with RIPA buffer (Tebu-Bio) in presence of antiproteases (Roche). The protein concentration was determined by the bicinchoninic acid method, according to the manufacturer's instructions (Pierce Chemical Co.). Bovine serum albumin (Pierce Chemical Co.) was used as standard. Cell lysates were resolved on SDS-PAGE and transferred to a Hybond-ECL membrane (GE Healthcare). Membranes were incubated with rabbit anti-CYP3A4 (5316, 1/10,000, Epitomics) or mouse anti-GAPDH (sc#32233, 1/5,000, Santa Cruz) monoclonal antibodies. Immunocomplexes were detected with horseradish peroxidase-conjugated rabbit (A0545, 1/10,000, Sigma) or mouse (A9044, 1/10,000, Sigma) secondary antibodies followed by enhanced chemiluminescence reaction (Millipore). Chemiluminescence was monitored using a ChemiDoc-XRS⁺ apparatus (Bio-Rad Laboratories).