Co-immunoprecipitation

To detect the endogenous interaction, cells were transfected with or without indicated plasmids for different experimental purpose and lysed in IP lysis buffer (Thermo) containing a protease inhibitor cocktail (Roche). The cell extracts were purified by centrifugation at 10,000 × g for 10 min at 4 °C. Then the supernatants were incubated with indicated antibodies or IgG control derived from the same species as the indicated antibody overnight at 4 °C, followed by incubation with Protein G magnetic beads (Thermo) for 2 h at 4 °C. The reaction mixtures were washed three times with IP lysis buffer supplemented with protease inhibitors, and harvested by centrifugation. After the immunoprecipitated proteins were denatured, the direct interactions between proteins were analyzed by immunoblotting performed according to standard protocols. The antibodies used for immunoprecipitation can be found in Supplementary Table ⁴.