Chemiluminescent hrp substrate

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).

Western blot was carried out as described previously by us [3 (link), 26 (link)]. Briefly, the total proteins were collected from RIPA cell lysis by centrifugation. The proteins were denatured and separated by SDS-PAGE and subsequently transmitted into 0.22μm PVDF membrane. The membrane was blocked with Protein Free Rapid Blocking Buffer (Epizyme, Shanghai, China) for 20 minutes and subjected for antibody incubation overnight at 4° C. The follow antibodies were applied, including incubated with Rabbit anti-Flotillin-2(C42A3) (#3436, dilution 1:1000, CST, MA, USA), rabbit anti-BCAT1 antibody (D121976, dilution 1:200, BBI, Shanghai, China), rabbit anti-c-Myc (A1309, dilution 1:500, Abclonal, Wuhan, China), rabbit anti-p-AKT (Ser473)(D9E) (#4060, dilution 1:1000, CST, MA, USA), rabbit anti-AKT (#9272, dilution 1:1000, CST, MA, USA), rabbit anti-p-NF-κB p65 (Ser536)(93H1) (#3033, dilution 1:1000, CST, MA, USA), rabbit anti-NF-κB p65(D14E12) (#8242, dilution 1:1000, CST, MA, USA), and rabbit anti-GAPDH (AB-P-R001, dilution 1:1000, Goodhere, Hanzhou, China). Next day, after incubated with anti-rabbit or anti-mouse IgG HRP-conjugated secondary antibodies (D110058 or D110098, dilution 1:3000, BBI, Shanghai, China), the protein levels were visualized by chemiluminescent HRP substrate (EpiZyme, Shanghai, China).

The Western blot (WB) was performed as previously described [16 (link)]. Briefly, RIPA (#WB3100, NCM Biotech, Suzhou, China) treated cell lysis was centrifuged at 4 °C to obtain total proteins. Proteins were denatured and electrophoresed on polyacrylamide gels with 12% sodium dodecyl sulfate before being transferred to polyvinylidene difluoride membranes. After blocking with 5% non-fat milk, the membrane was incubated with the primary antibodies, including rabbit anti-FBXO43, rabbit anti-IGF2BP2, anti-METTL3 (dilution: 1:500, 1:1000, 1:500; #55176-1-AP, #11601-1-AP, #15073-1-AP, Proteintech, IL, USA), rabbit anti-UBE2C and anti-p21 (dilution: 1:400, #D124141, #D120403, BBI, Shanghai, China), mouse-p53 (dilution: 1:100, #sc-126, Santa Cruz, TX, USA), rabbit-Cleaved-PARP-1 (dilution: 1:500, Immunoway, TX, USA) and rabbit anti-GAPDH antibody (dilution: 1:2000, #BS65656, Bioworld, Nanjing, China), respectively. After incubation with HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (dilution: 1:3000, #D110058/D110098 BBI, Shanghai, China), the protein amounts were visualized using chemiluminescent HRP substrate (#SQ202, EpiZyme, Shanghai, China). The optical densities of Western blot bands were obtained using ImageJ analysis software.