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Immunoblotting Cell extracts were prepared in lysis buffer (50 mM Tris-HCl pH 6.8, 120 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM PMSF, 1 mM -mercaptoethanol, 1 mM Na 3 VO 4 , 1 mM NaF, and 1 g/ml aprotinin) for 30 min on ice, and centrifuged at 15,000 g for 15 min to remove insoluble materials