Ddh2o

Loading gel was prepared as follows 30% acrylamide, 0.5 mM Tris (pH 6.8), double distilled water (dd.H₂O), 10% SDS, ammonium persulfate, and tetramethylethylenediamine while the separating gel was also prepared in the same manner with 1.5 mM Tris at pH 8.8 (BIO-RAD; CA, USA). Gel electrophoresis was performed in a running buffer (×10, Tris/Glycine/SDS Buffer; BIO-RAD, CA, USA). Proteins (20 µg/lane) were separated by 10% SDS-PAGE and transferred in transfer buffer Tris 25 mM, glycine 192 mM, 0.01% SDS and 10% methanol and dd.H₂O (Millipore) onto polyvinylidene difluoride membranes. The separated proteins were pre-incubated with 5% nonfat in Tris buffered saline (Tris, sodium chloride, dd.H₂O and Tween-20) and incubated overnight at room temperature. Membranes were then probed with the following primary antibodies against protein kinase B (PKB)/Akt (1:1000), p-PKB/Akt at Ser473 (1:2000), p38 MAPK (1:1000), poly adenosine diphosphate ribose polymerase (PARP) (1:1000), cleaved PARP (1:1000), Caspase 3 (1:1000), and cleaved Caspase 3 (Asp 175) (1:1000). All primary antibody probes were purchased from Cell Signaling Technology, Danvers, MA, USA. And then, the membranes were incubated with horseradish peroxidase-conjugated secondary anti-rabbit immunoglobulin G (1:500; Santa Cruz Biotechnology, CA, USA).