2.5. Western Blot Analysis

SH-SY5Y cells were seeded in the 10 cm Petri dishes with a density of 5 × 10⁵ cells per dish. Firstly, the cells were pretreated with increasing concentrations (0.37, 1.1, and 3.3 μg/mL) of Triphala for 24 h and stimulated with 400 μmol/L H₂O₂. The blank control and model control were established as described above. Then, the cultured cells were harvested by centrifugation and fractionated using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology Inc., Nantong, China) following the manufacturer's instruction with the supplement of protease inhibitor cocktail and phosphatase inhibitor cocktail, offered by Sigma-Aldrich Corp. (St. Louis, MO). The protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). The total or nuclear proteins were separated by SDS-PAGE electrophoresis, transferred to a nitrocellulose membrane, and then incubated with monoclonal antibodies, including phospho-p44/42 mitogen-activated protein kinase (MAPK) extracellular signal-related kinase (ERK)1/2. (Thermo Fisher), phospho-c-Jun amino-terminal kinases (JNK)1/2 (Thermo Fisher), and phospho-p38 MAPK, superoxide dismutase 1 (SOD1), and catalase, which were purchased from Cell Signaling Technology (Danvers, USA). Afterwards, rabbit anti-mouse secondary antibody (Abcam, Cambridge, UK) and goat anti-rabbit secondary antibody (Abcam) were attached and expression levels of proteins were detected by chemiluminescence using a Pierce ECL Plus Western Blotting Substrate (Thermo Fisher).