4.6. Western Blot Analysis

After MPP⁺- and VPA treatment for 48 h, DA neuron cultures were washed with ice-cold PBS for three times and lysed with a lysis buffer (Cell Signaling Technology Inc., Beverly, MA, USA). The protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific Inc.). Equal amounts of protein (60 μg/sample) were loaded and separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific Inc.). Membranes were blocked with 5% nonfat milk solution in Tris-buffered saline with 0.1% Triton X-100 (TBST) for 1 h at room temperature and then incubated overnight at 4°C with the primary antibody dilutions in TBST: Akt (1 : 1000), phospho-Akt (1 : 800), ERK1/2 (1 : 1000), phospho-ERK1/2 (1 : 800), GSK3β (1 : 1000), and phospho-GSK3β (pSer⁹) (1 : 800; all from Cell Signaling Technology Inc.), Bax (1 : 800), Bcl-2 (1 : 1000), cytochrome c (1 : 1000), caspase-3 (1 : 800), and caspase-9 (1 : 800; all from Cell Signaling Technology Inc.). After that the membranes were washed and incubated with secondary antibodies for 1 h at room temperature. Immunoreactivity was detected with Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA). Image J image analysis software (National Institute of Heath, Bethesda, MD, USA) was used to quantify the optical density of each band. The activation of Akt and ERK1/2 is presented as the ratio of phosphorylated kinase bands to total kinase bands. The activation of caspase-9 and caspase-3 is presented as the ratio of cleaved bands to the total bands.