Cellular Fractionation and Lysis Protocol

Cells were lysed and assayed as previously reported [10 (link)]. Briefly, cells were lysed in cytoplasm lysis buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.2 mM EDTA, 1 mM DTT), containing protease inhibitors, 0.5 mM phenylmethylsulfonylfluoride (PMSF) and 0.6% Nonidet P-40 (Sigma Chemical Co., St Louis, MO, USA). Lysates were centrifuged at 10.000 rpm 10 min at 4°C and the supernatants (cytoplasmic fractions) were split into aliquots and rapidly frozen. The nuclear pellet was washed in buffer A without Nonidet P-40 and finally resuspended in nuclear lysis buffer B (20 mM HEPES pH 7.9, 0.4 M NaCl, 2 mM EDTA, 1 mM DTT), containing protease inhibitors and 1 mM PMSF (Sigma Chemical Co., St Louis, MO, USA). Samples were incubated on ice 30 min and centrifuged at 13.000 rpm 10 min at 4°C; the supernatants (nuclear fractions) were split into aliquots and rapidly frozen or used for western blot analysis.