Protein Extraction and Western Blot Protocol

The total proteins extraction and western blot were performed as our previous description^{29 (link),32 (link)}. In brief, the cells were disrupted by radioimmunoprecipitation assay lysis buffer and the total proteins were collected by centrifugation. The denatured proteins (30 μg per lane) were separated by sodium dodecyl sulfate poly-acrylamide gel electrophoresis and subsequently transmitted into 0.22 μm polyvinylidene difluoride membrane. After blocked with 5% nonfat milk at room temperature for 1 h, the membrane was incubated with rabbit anti-RKIP antibody (D221145, dilution 1:500, BBI, Shanghai, China), rabbit anti-NRF2 antibody (16396-1-AP, dilution 1:50, Proteintech, IL, USA), rabbit anti-NQO1 antibody (D261049, dilution 1:500, BBI, Shanghai, China), rabbit anti-HO-1, rabbit anti-GCLC (D123963, dilution 1:300, BBI, Shanghai, China), and mouse anti-GAPDH antibody (AC002, dilution 1:1000, Abclonal, Wuhan, China), respectively, overnight at 4 °C. Next, after incubated with anti-rabbit or anti-mouse IgG HRP-conjugated secondary antibodies (D110058 or D110098, dilution 1:3000, BBI, Shanghai, China), the protein amounts were visualized by chemiluminescent HRP substrate (EpiZyme, Shanghai, China).