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Preparation of cell extracts and western blot analysis Cells were trypsinized, washed with PBS and resuspended in lysis buffer [50 mM TrisHCl (pH 7.5), 120 mM NaCl, 0.5% (v/v) Nonidet P-40] supplemented with protease inhibitor cocktail (Roche, 11873580001) and incubated for 5 min on ice

Detection methods that did not exist five years ago are now routine