Enhanced chemiluminescence western blot kit

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).

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).

Cells were washed with cold PBS and lysed on ice in 50 μL of lysis buffer (50 mM Tris-HCl, 1 mM EGTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, pH 7.5). Lysates were centrifuged at 10,000 × g for 15 min at 4 °C, and the supernatant fractions were collected. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to an Immobilon-P membrane (GE Healthcare Life Science, Pittsburgh, PO, USA). Specific proteins were detected using an enhanced chemiluminescence Western blot kit (EMD Millipore, Darmstadt, Germany), according to the manufacturer’s instructions.