Supersignal west dura hrp detection kit

After treatment, the cells were detached by trypsinization, collected by centrifugation, and then lysed in the PBS lysis buffer containing protease and phosphatase inhibitor cocktail (Sigma-Aldrich). The cell lysates were centrifuged at 20,000 x g at 4 °C for 10 min, and then the supernatant was collected and used as a crude extract. The protein concentration in the extracts was assessed by Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Extracts containing equal protein (30 µg) were separated by electrophoresis on an SDS-polyacrylamide gel. The electrophoresed proteins were transferred to a nitrocellulose membrane (PROTRAN BA85, 0.45 μm; Sigma) with a Transphor Unit (Bio-Rad Laboratories, Hercules, CA, USA). The transferred membrane was blocked with 1% (w/v) BSA in PBS followed by a 1-h incubation with the primary antibodies and then the secondary antibodies. The signal was developed with ECL chemiluminescence reagent (SuperSignal West Dura HRP Detection Kit; Pierce Biotechnology, Rockford, IL, USA), and the signals were acquired and quantitated with an image analysis system (Fujifilm, Tokyo, Japan).

Cells were lysed using RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Sigma-Aldrich) at 4 °C for 30 min. After centrifugation (20,000× g, 4 °C, 15 min) to remove insoluble debris, the supernatant (crude protein extracts) was collected for the following analysis. Protein concentration was assessed by Bradford method in accordance with the manufacturer’s instruction (Bio-Rad Laboratories, Hercules, CA, USA). Crude proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred onto a polyvinylidene difluoride membrane (Immobilon, Merck-Millipore, Bedford, MA, USA), and then blocked with 3% (w/v) BSA/PBS at 25 °C for 1 h. Thereafter, the membrane was incubated with primary antibodies (1000-fold dilution) for 2 h and then incubated with secondary antibodies (2000-fold dilution) for 2 h after being washed with 0.5% Tween-20/PBS. The antibody–antigen complex was detected using an ECL chemiluminescence reagent (SuperSignal West Dura HRP Detection Kit; Pierce Biotechnology, Rockford, IL, USA), and the resulting signals were acquired and semi-quantitated using an image analysis system (Fujifilm, Tokyo, Japan). Signals from PBS treatment were used as control.