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 incubated with 5% nonfat milk for 30 min and then overnight with the primary antibodies. After overnight incubation, the membranes were incubated with secondary antibodies for 2 h, and were exposed using an enhanced chemiluminescence Western blot kit (EMD Millipore, Darmstadt, Germany). All protein band intensity was measured using ImageJ. For apoptosis analysis, cells were fixed with 100% ethanol for 2 h at 4 °C. Next, cells were incubated with RNase for 30 min at 37 °C and stained with propidium iodide. DNA content was measured by flow cytometry (BD Biosciences).