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Western blotting Cells were washed once with cold PBS and lysed in RIPA buffer (50 mM Tris-HCl pH 7.5, 125 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, 1mM Na-fluoride, 1 mM Na orthovanadate, phenylmethanesulfonylfluoride [PMSF], and 1% protease inhibitor cocktail), vortexed, and incubated on ice 20