Protein Extraction and Immunoblotting Protocol

The cell pellets were lysed with 1 % NP-40 lysis buffer (20 mmol/L Tris–HCl, pH 7.5; 1 mmol/L EDTA; 150 mmol/L NaCl; 1 % NP-40) phosphatase inhibitors cocktail 2 and 3 (Sigma) and containing protease inhibitors (Roche) for 30 to 45 min at 4 °C. Protein lysates were prepared after calculating protein concentration using the Bradford reagent (Biorad) and 50 μg of protein was resolved on 10 % SDS-PAGE followed by transferring to nitrocellulose (Millipore) [30 (link)]. Immunoblotting was performed with primary antibodies against BCL-xL (Santa Cruz Biotechnology) and β-actin (Sigma). The secondary anti-mouse antibodies were purchased from Thermo-Fisher Scientific.