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The plates were then washed twice with ice-cold PBS, scraped, pelleted 250 g at 4 C for 3 min and lysed with RIPA buffer (50 mM Tris-Cl (pH = 8), 150 mM NaCl, 5 mM EDTA, 1 % NP-40, 0.5 % sodium deoxycholate, 0.1% SDS, 1x protease and 1x phosphatase inhibitors)