Samples of the SNpc were lysed in RIPA lysis buffer containing protease inhibitor and phosphatase inhibitor cocktail (Beyotime, Shanghai, China). The protein concentration was determined using BCA kits (Beyotime, Shanghai, China) (Zhang et al., 2020). For Western blotting, the samples were boiled in 5 × loading buffer (Applygen, Beijing, China), electrophoresed on a 12% Tris-glycine gel by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride membranes with a pore size of 0.45 μm (Merck Millipore, MA, United States) (Schägger, 2006). After blocking with 7% non-fat milk at room temperature for 2 h, the membranes were incubated for 24 h with primary antibody overnight at 4°C and then with secondary antibodies coupled to horseradish peroxidase for 2 h. The following primary antibodies purchased from Cell Signaling Technology (Boston, MA, United States) were used for Western blot analysis: phospho-ERK1/2 (1:1,000), caspase-3 (1:1,000), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1,000). The following secondary antibodies were used for Western blot analysis: anti-rabbit IgG-HRP (1:10,000) and anti-mouse IgG-HRP (1:10,000) (Santa Cruz Biotechnology, Dallas, TX, United States). Cross-reactivity was visualized using ECL Western blot detection reagents (Millipore, Burlington, MA, United States), analyzed by scanning densitometry using UVP VisionWorksTM LS Software (UVP, Cambridge, United Kingdom) and quantified with ImageJ Software (Zhang et al., 2020).

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