Protein Extraction and Western Blotting

Western blotting and IP assay were conducted following the protocols [34 (link)]. In brief, the cells or tissues were lysed in RIPA lysis buffer (Beyotime, P0013B) with Protease Inhibitor Cocktail (Roche, 05892970001) and Phosphatase Inhibitor (Roche, 04906845001) for 1 h. After 12,000 rpm centrifugation for 20 min at 4 °C, the supernatant was taken. Then, the protein supernatants were separated using SDS-PAGE and transformed onto nitrocellulose filter membranes (Pall Corporation, 66485). After blocking by 5% BSA for 1 h, the membranes were incubated with primary antibodies at 4 ℃ overnight. The blots were measured by laser scanner (Odyssey, Licor, USA). Cells were lysed in western blot & IP lysis buffer (Beyotime, P0013). The lysates were incubated with protein A/G beads (Santa Cruz, sc-2002) and primary antibody for 12 h at 4 °C. The precipitates were collected by centrifugation at 1500 rpm for 5 min at 4 °C and washed three times with the cold 1×TBS. The protein samples were subjected to western blotting analysis.