P0222

The proteins were separated by 12.5% SDS-PAGE (EpiZyme; PG113) at a constant voltage of 100 V at room temperature for 1.5 h. Separated proteins were then blotted onto PVDF membranes (Bio-Rad; 1620177) at a constant current of 300 mA for 1 h. Membranes were blocked with quick block solution (Beyotime Biotechnology; P0222) for 15 min, then incubated with primary antibodies against caspase 3 (1:1000; Cell Signaling Technology), cleaved caspase 3 (1:1000; Cell Signaling Technology) and GADD45α (1:500; BOSTER Biological Technology) at 4 °C overnight. After washing, the membranes were then incubated with HRP-labeled 1:5000 diluted goat anti-rabbit (ZSGB Bio.; ZB-2301) or anti-mouse (ZSGB Bio.; ZB-2305) secondary antibodies at room temperature for 1 h. Finally, membranes were visualized with an enhanced chemiluminescence detection system (Tanon; 5200CE).

Grinding adjacent normal tissues and ccRCC tissues were washed in PBS, collected with a cell scraper and ultracentrifuged for 20 min at 10,000 × g at 4°C. The sediments were resuspended in high efficiency cell tissue rapid lysis buffer (RIPA; P0013J, Beyotime, China) with 1% phenylmethanesulfonylfluoride proteinase inhibitors (PMSF; P1008, Beyotime) and 1% phosphatase inhibitors (C071, ChemDiv, USA) inside RIPA. Subsequently, the mixtures were boiled at 95°C for 10 min and then were preserved at −80°C if necessary. Protein quantification were implemented by utilizing the BCA protein assay kit (P0012, Beyotime, China). Total protein mixtures of 20 μg were electrophoresed by direct current in SDS-PAGE gels (LK102, Epizyme Biotech, China) using a Miniprotein III machine (Bio-Rad, USA) and were electro-transferred to PVDF membranes (Millipore, USA) for 2 h, followed by overnight application with primary antibodies targeted CD45RO (5 μg/ml; ab23, Abcam) and GAPDH (1/10000; ab181602, Abcam) at 4°C. Then the membranes were rinsed in triplicates with PBST solution (P0222, Beyotime) and were applied with the secondary goat anti-rabbit IgG antibodies (1:5000; Bioworld Technology, USA) at 20°C for 1 h. Subsequently, the membranes were rinsed again in triplicates and prepared for chemiluminescence by utilizing the Immobilon Western Chemiluminescent HRP Substrate Kit (Millipore).