Mouse anti bak

Cells were collected and resuspended in RIPA lysis buffer for 60 min incubated on ice. After centrifuging for 20 min at 12000g and 4°C, the supernatant was collected. Protein concentrations were determined by using Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific, Rockford, IL, USA). 60 μg of protein was separated on a 10% SDS-PAGE gel and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was rinsed with TBST and incubated in 5% nonfat milk diluted with TBST at 37°C for 2 h. The membrane was then incubated with mouse anti-Bak (1:1000 dilution, Cell signaling technology), mouse anti-c-PARP (1:1000 dilution, Cell signaling technology), and mouse anti-GAPDH primary antibodies (1:3000 dilution, Santa Cruz, Dallas, Texas, USA) respectively overnight at 4°C. After washing with TBST 3 times for 15 min each time, the membrane was incubated with the appropriate secondary antibodies for 1 hour at 37°C. The membranes were then washed with TBST 4 times for 10 min each time and visualized the bands by using an ECL kit (Millipore, Billerica, MA) in a Gel Doc™ XR+ and ChemiDoc™ XRS+ System (BioRad, Hercules, CA). Image-Pro plus software 6.0 (Media Cybernetics, Rockville, MD, USA) was used to analyze the relative protein expression which was normalized to GAPDH.