4.3. Cell Fractionation

To obtain the cytoplasmic extract, cells (8.5 × 10⁶ per extract) were washed with PBS, resuspended in 100 μL of a 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.6) buffer containing 60 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.075% (v/v) NP-40, 1 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF) and centrifuged at 400× g for 4 min at 4 °C. To obtain the nuclear extract, the pellet remaining after centrifugation was washed with a 100 μL 10 mM HEPES (pH 7.6) buffer without detergent and resuspended in 100 μL of a lysis buffer (420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 1 mM PMSF and 25% (v/v) glycerol, 20 mM tris(hydroksymethyl)aminomethane (Tris)-HCl, pH 8.0) supplemented with the protease and phosphatase inhibitors. Samples were cleared by centrifugation at 16,000× g for 10 min at 4 °C.