Cell lysate is prepared in modified RIPA buffer (50mMTris, pH7.2, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1% NP-40, 1mM Na3VO4, 5mM NaF, 400µM PMSF, 1µg/ml of pepstatin A, 5µg/ml of aprotinin, 5µg/ml of leupeptin). Cell debris is removed by centrifugation, and the protein concentration is determined with the Bio-Rad protein assay. The cell lysate is diluted and boiled for 5 min with nonreducing sample buffer (50 mM Tris-HCl, pH 6.8, 5% glycerol, 1% SDS, 0.002% bromphenol blue) containing 5% beta-mercaptoethanol. (SC2706)