Phosphorylation protease inhibitor

RIPA lysis buffer (Servicebio, China) with phosphorylation protease inhibitor (Servicebio, China) were used to extract the protein of cells. Protein A-sepharose beads were washed by RIPA and centrifuged at 1,000 × g in a micro centrifuge to remove supernatant. Cell lysis buffer was mixed with beads and primary antibodies against the specific antigen were added. The mix was incubated with rotation overnight at 4 °C to form the immunocomplex and the step proceeded for Western blotting assay. Total proteins (input controls) and normal rabbit IgG controls were simultaneously assayed. The antibodies used for IP were listed in the Supplementary Table 2.

RIPA lysis buffer (Servicebio, China) with phosphorylation protease inhibitor (Servicebio, China) were used to extract the protein of cells. The proteins were separated by gel electrophoresis and transferred to polyvinylidene fluoride membranes (Roche, Basel, Switzerland). Membranes were blocked with 5% nonfat milk or bovine serum albumin (BSA) for 2 hours at room temperature and incubated with primary antibodies in 4 °C overnight. The membranes were washed and then incubated with secondary antibodies for 2 hours. The antibodies used for Western blotting were listed in the Supplementary Table 2.