2.9. Western Blot Analysis

H9C2 cells were supplemented with Carex and incubated with H₂O₂ to induce oxidative stress. Cells were lysed in RIPA buffer, and the protein concentration was measured, using the BCA assay. Forty micrograms of protein were separated by SDS-PAGE. The proteins were then transferred to a PVDF membrane at 70 V for 2 h. After blocking with 5% skim milk, the membranes were then incubated with Nrf-2 (Cell Signaling Technology, MA, USA), HO-1 (Cell Signaling Technology, MA, USA), and GAPDH (Cell Signaling Technology, MA, USA) antibodies overnight at 4 °C. After washing, an anti-rabbit IgG with horseradish peroxidase (Cell Signaling Technology, USA) was incubated. ECL Blotting Reagent (Cytiva, Marlborough, MA, USA) was used for the chemiluminescence reaction, followed by analysis using the ChemiDoc™ XRS System (Bio-Rad, Hercules, CA, USA).