The cells were swollen in hypotonic buffer (10 mM Tris-Cl [pH 7.5], 10 mM KCl, 0.5 mM EGTA, 1.5 mM MgCl 2 , 1 mM PMSF, and 13 EDTA-free protease cocktail) for 2.5 min at 4 C.