Cell Lysis and Immunoprecipitation Protocol

HEK293T cells were transiently transfected using polyethylenimine. Sixteen hours after transfection HEK293T cells were incubated with or without MG132 for 3 h before collection. NHFs, NIH3T3, RPE and U2OS cells were subjected to experimental conditions (serum-starvation before re-addition of serum or siRNA) for various times before collection. Cell lysis was carried out with lysis buffer (50 mM Tris, at pH 8.0, 150 mM NaCl, glycerol 10%, 1 mM EDTA, 50 mM NaF and NP-40 0.1%) supplemented with protease and phosphatase inhibitors. For denaturating conditions, cells were lysed in lysis buffer containing 1% SDS, 5 mM dithiothreitol, 1 mM sodium vanadate and 50 mM Tris at pH 7.4 and boiled for 10 min before immunoprecipitation. Immunoprecipitation and immunoblotting were performed as previously described^{49 (link),50 (link)}.