Ecl prime western blotting detection system

Human samples were dissociated and lysed in radioimmunoprecipitation (RIPA) buffer supplemented with protease inhibitor cocktail (Epizyme Biotech, China). Its concentration was measured with a BCA protein assay kit (Takara Bio, Tokyo, Japan). Protein samples were separated by electrophoresis on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with skim milk for 1 h in Tris-buffered saline with Tween 20 (TBST), the membranes were incubated with the primary antibodies including CDKN2A (#AF5484, Affinity, 1:1,000), SDHB (#ab175225, Abcam, 1:1,000), CCS (#DF3971, Affinity, 1:1,000), ULK1 (#DF7588, affinity, 1:1,000), CMC1 (#24030-1-AP, Proteintech, 1:1,000), and ACTIN (#3700, CST, 1:1,000) at 4℃ overnight. The membranes were washed three times with TBST, 10 min each time, and subsequently incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (#7074, CST, Boston, MA, USA, 1:5,000) for 1 h. Protein bands were visualized using the ECL Prime Western Blotting Detection System (32209, Thermo Fisher Scientific, Waltham, MA, USA).

Cells were harvested and lysed in RIPA buffer supplemented with protease inhibitor cocktail (Epizyme Biotech). Protein concentration was determined with a BCA protein assay kit (Takara Bio, Tokyo, Japan). Protein samples were separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) gels and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with skim milk for 1 hour in TBST, the membranes were incubated with the primary antibodies included anti-PDL1 (#1-76769, Novus, USA, 1 : 1000), anti-PDL1 (#DF6526, affinity, 1 : 1000), anti-HSP90 (#4877, CST, Boston, MA, USA, 1 : 1000), anti-IFNα (#DF6086, affinity, 1 : 1000), anti-IFNβ (#ab218229, Abcam, 1 : 1000), Anti-IFNγ (#DF6045, affinity, 1 : 1000), anti-JNK2 (#9258, CST, 1 : 1000), P-JNK (#4668, CST, 1 : 1000), P-c-fos (#5348, CST, 1 : 1000), and P-c-jun (#2361, CST, 1 : 1000) at 4°C overnight. The membranes were washed three times with TBST, 10 min each time, and subsequently incubated with horseradish peroxidase- (HRP-) conjugated secondary antibody (#7074, CST, Boston, MA, USA, 1 : 5000) for 1 h. Protein bands were visualized using the ECL Prime Western Blotting Detection System (32209, Thermo Fisher Scientific, Waltham, MA, USA).