The protocol used was modified from a previously described method (57). HeLa cells (2 × 106) were plated in a 10-cm dish ~16 hours before the experiment. After drug treatment, cells were washed once with 5 ml of PBS, scraped in PBS, and pelleted at 350g for 5 min at 4°C. To lyse cells, pellets were resuspended in 50 μl of NETN buffer [100 mM NaCl, 20 mM tris-HCl (pH 7.5), 0.5% NP-40, and 2 mM EDTA] for every 5 × 105 cells and incubated for 15 min on ice during which they were vortexed twice. The nuclei were pelleted in a microcentrifuge at 14,000 rpm for 5 min. The supernatant was moved to a new tube and, after another centrifugation at 14,000 rpm for 5 min, was saved as the cytoplasmic/soluble nuclear protein fraction. The pellet was washed once with 50 μl of NETN buffer, resuspended in 50 μl of nuclear digestion buffer [20 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM CaCl2, and micrococcal nuclease (200 U/ml)], and incubated for 8 min at 37°C. This pellet was saved as the chromatin fraction. After adding 10 μl of 6× SDS sample buffer with BME, the samples were boiled for 5 min and clarified in a microcentrifuge at 13,000 rpm for 10 min at room temperature, and 15 μl of the sample was loaded for SDS-PAGE and Western blot.

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