Subcellular Fractionation
Subcellular fractionation was performed by differential centrifugation as previously described6. Briefly, cells were resuspended in ice-cold Buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, 0.4 % NP-40, 0.5 mM PMSF, complete protease inhibitor cocktail) at 4 °C for 5 min. Lysates were centrifuged at 4 °C, 500 × g for 3 min, and supernatants were collected as cytosolic fractions. Pellets were incubated in Buffer C (20 mM HEPES pH7.9, 420 mM NaCl, 1.5 mM MgCl2, 25% glycerol, 0.5 mM PMSF, 0.2 mM EDTA, 0.5 mM DTT, complete protease inhibitor cocktail) at 4 °C for 10 min. Supernatants were collected as nuclear fractions following a centrifuge at 4 °C, 13,800 × g for 10 min.