For immunoprecipitation experiments, cells were lysed using an IP lysis buffer containing 25 mM HEPES, pH7.4, 1 mM EDTA, 10 mM NaCl, 0.5% Triton X-100, protease inhibitors cocktail (Roche) and 1 mM PMSF, except for the ubiquitination assay in which the IP lysis buffer contains 0.2% SDS (Extended Data Fig. 4e). Equal amount of protein was incubated overnight with Fast Flow Protein G agarose beads (Millipore) and mouse IgG or specific antibody in IP lysis buffer. After pull-down, protein-G beads were washed five times with IP washing buffer (25 mM HEPES, pH 7.4, 1 mM EDTA, 100 mM NaCl, 0.5% Triton X-100 and protease inhibitors cocktail (Roche) and boiled with 2×SDS sample buffer (Bio-Rad) containing 5% β-mercaptoethanol (Sigma). Western blotting was then carried out with primary antibodies listed in Supplementary Table 1b.
