Western Blot and Apoptosis Analysis

Total lysates were obtained using modified RIPA lysis buffer as described previously [41 (link),42 (link),43 (link)]. The proteins were separated by SDS-PAGE and transferred onto an Immobilon-P membrane (GE Healthcare Life Science, Pittsburgh, PO, USA). The membranes were exposed using an enhanced chemiluminescence Western blot kit (EMD Millipore, Darmstadt, Germany). For apoptosis analysis, cells were harvested and fixed with 100 % ethanol for 2 h at 4 °C. Then cells were resuspended in 50 μg/mL RNase for 30 min at 37 °C, and added to 50 μg/mL propidium podide. The stained cells were analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA).