Immobilon ecl kit

Cells were lysed in ice-cold RIPA lysis buffer (50 mM Tris–HCl, pH = 7.4, 1 mM EDTA, 150 mM NaCl, 0.5% NP-40, 1 mM sodium vanadate, 0.5% sodium deoxycholate, 0.05% SDS) containing 1× EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Total protein was extracted from cell lysates by centrifugation (14 000 g) at 4 °C for 10 min and quantified using a BCA Protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) following the protocols of manufacturers. Protein extracts were resolved on a 10% SDS polyacrylamide gel and blotted onto a Hybond-P PVDF membrane (Amersham Bioscience, Piscataway, NJ, USA). The following primary antibodies were used: anti-Bcl-2 (Abcam, Cambridge, UK; dilution 1 : 1000), anti-Bax (Cell Signaling Technology, Danvers, MA, USA; dilution 1 : 1000) and anti-β-actin (Abcam; dilution 1 : 2000). Horseradish peroxidase-conjugated IgG antibody (Abcam; dilution 1 : 5000) was used as a secondary antibody. Protein bands were visualized using the immobilon ECL kit (Millipore, Billerica, MA, USA) and band intensities were quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Cells were lysed with cell lysis buffer (Cell Signaling Technology) supplemented with protease inhibitor “cocktail” (Roche). Protein concentrations in the extracts were measured by BCA assay (Pierce). Overall, 40 μg proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by electrotransfer to polyvinylidene difluoride membrane (Hybond‐P; GE Healthcare Life Sciences). Membranes were probed using indicated antibodies against targeted proteins, followed by the HRP‐conjugated second antibody (Cell Signaling Technology, 1:10,000). Bands were revealed with Immobilon ECL kit (Millipore) and recorded on X‐ray films (Kodak, Xiamen, China).