Be0101 100

Cells were lysed at 48 h post-transfection with BROD (Thermo Fisher Scientific) buffer for 30 min on ice, then denatured with loading buffer boiled at 100°C for 10 min. Protein samples were separated by SDS-PAGE gel electrophoresis and transferred onto a PVDF membrane. Membranes were blocked with 5% skim milk at 37°C for 1 h, then incubated with primary antibodies at 4°C overnight. Primary antibodies included KDM5B (A7772, 1:1000, ABclonal, Wuhan, Hubei, China) and GAPDH (CW0100M, 1:5000, CWBIO, Nanjing, Jiangsu, China). After rinsing three times in TBST, the membranes were incubated at 37°C for 1 h with HRP-conjugated secondary antibodies (BE0101-100 or BE0102-100 Easybio, Beijing, China). After rinsing three times in TBST, membranes were treated with super ECL Plus Western Blotting Substrate (Thermo Fisher Scientific) and signals were visualized using a Tanon 5200 (Tanon Science & Technology Co. Ltd, Shanghai, China).