Subcellular Fractionation

Subcellular fractionation was performed as previously described [22]. Briefly, cells were washed 3× with PBS, harvested and allowed to pre-swell for 10 min at 4 °C in extraction buffer. Then the pelleted cells were lysed for 10 min on ice with complete extraction buffer (10 mM KCl, 10 mM Na-HEPES, pH 7.6, 1.5 mM MgCl₂, 0.1% Nonidet-P40, 0.5 mM dithiothreitol, 0.5 mM spermidine, 0.15 mM spermine, 1 mM phenylmethylsulphonyl fluoride, 1× protease inhibitor cocktail (Roche, Laval, Qc, Canada)). Lysates were centrifuged (2 min, 6000×g) and supernatant reserved as cytosolic fraction. The nuclear pellet was collected and washed 3× with wash buffer (50 mM NaCl, 20 mM Na-HEPES, pH 7.6, 25% glycerin, 0.2 mM EDTA, 1.5 mM MgCl₂, 0.5 mM dithiothreitol, 0.5 mM spermidine, 0.15 mM spermine, 1 mM phenylmethylsulphonyl fluoride, 1× protease inhibitor cocktail (Roche)). The washed pellets were lysed in nuclear extraction buffer (500 mM NaCl, 20 mM Na-HEPES, pH 7.6, 25% glycerin, 0.2 mM EDTA, 1.5 mM MgCl₂, 0.5 mM dithiothreitol, 0.5 mM spermidine, 0.15 mM spermine, 1 mM phenylmethylsulphonyl fluoride, 1× protease inhibitor cocktail) for 30 min on ice. Fractions were verified for lack of cross-contamination between nuclear and nonnuclear compartments by Western blot (data not shown).