Western Blot Analysis of Protein Expression

Proteins were extracted as previously described [19 (link)]. Subsequently, SDS-PAGE (7.5–10% polyacrylamide gels) was used to separate the proteins, which were then transferred to polyvinylidene fluoride (PVDF) membranes (Thermo Scientific). The membranes were blocked with 5% skim milk and then incubated with the primary antibodies (1:1000; CST, Danvers, MA, USA) overnight, followed by incubation with secondary antibodies (1:2000; CST, Danvers, MA, USA) at room temperature. The immunoreactive protein bands were visualized using an enhanced chemiluminescence (ECL) kit (Thermo Scientific). A Luminescent Image Analyzer LAS4000 (Fuji Film, Tokyo, Japan) and ImageJ software (NIH, Bethesda, MD, USA) were used to detect and quantify the protein band signals. U0126 ERK pathway inhibitor was obtained from Sigma-Aldrich (Danvers, MA, USA). ML385 NRF2 pathway inhibitor was purchased from MedChemExpress (Shanghai, China).