Western Blot Analysis of Protein Targets

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).