After 72 hours of transfection, cells were harvested and lysed in RIPA buffer mixed with 1% protease inhibitor mixture at 4°C for 30 minutes. The protein concentration was determined by the BCA method. The protein samples (40 μg) were divided by SDS‐PAGE and electrotransferred to PVDF membranes. Next, the membranes were blocked with 5% non‐fat milk for 2 hours and incubated overnight at 4°C with primary antibodies: RCC1 (1:1000, Abcam, USA), p27kip1 (1:1000, Abcam, USA), CDK4 (1:1000, ABclonal, China), PD‐L1 (1:1000, Abcam, USA), p‐RbSer780 (1:1000, Abways, China) and GAPDH (1:1000, Abcam, USA). Then, we washed the membranes with TBST buffer three times and further incubated with secondary antibodies (1:10 000, Immunoway, USA) for 1 hours at 25°C. Finally, the membranes were washed three times by TBST and freshly prepared ECL fluid (Beyotime Institute of Biotechnology, China) was applied for colour development in a dark room. The grey intensity analysis of western blotting images was carried out by ImageJ software.

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