Cell Lysis and Immunoprecipitation Protocol

Cells were lysed in EBC buffer (50 mM Tris, pH 7.5, 120 mM NaCl, 0.5% NP-40) or Triton X-100 buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100) supplemented with protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (phosphatase inhibitor cocktail set I and II, Calbiochem). The protein concentrations of whole cell lysates were measured by NanoDrop OneC using the Bio-Rad protein assay reagent according to manufacture instructions^{13 (link)}. Equal amounts of whole cell lysates were resolved by SDS-PAGE and immunoblotted with indicated antibodies. For immunoprecipitations analysis, 1000 μg lysates were incubated with the indicated antibody (1–2 μg) for 3–4 h at 4 °C followed by 1 h incubation with 10 μl Protein A magnetic beads (New England Biolabs). Or 1000 μg lysates containing tagged molecules were incubated with agarose beads coupled antibodies for the specific tag for 3–4 h at 4 °C. The recovered immuno-complexes were washed five times with NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA and 0.5% NP-40) before being resolved by SDS-PAGE and immunoblotted with indicated antibodies. Uncropped images are provided in Supplementary Fig. 14.