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Immunoblotting Cell and tissue lysates were prepared in RIPA lysis buffer (150 mM NaCl, 1% NP40, 0.5% DOC, 50 mM Tris-HCl at pH 8, 0.1% SDS, 10% glycerol, 5 mM EDTA, 20 mM NaF and 1 mM Na3VO4) supplemented with 1 mg/ml each of pepstatin, leupeptin, aprotinin and 200mg/ml phenyl-methyl-sulfonyl-fluoride (PMSF)