Cells (2 × 106) were washed with cold phosphate-buffered saline (PBS) and resuspended in 150 μl of hypotonic buffer [20 mM tris-HCl (pH7.4), 10 mM NaCl, and 3 mM MgCl2] supplemented with protease inhibitor cocktail for 15 min on ice. NP-40 detergent (0.5%) was then added, and the sample was vortexed for 10 s, followed by centrifugation for 10 min at 3000 rpm at 4°C. The supernatant was collected as the cytoplasmic fraction. The nuclear pellet was washed and resuspended in 150 μl of complete cell extraction buffer [10 mM tris (pH 7.4), 2 mM Na3VO4, 100 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol, 1 mM EGTA, 0.1% SDS, 1 mM NaF, 0.5% deoxycholate, and 20 mM Na4P2O7] supplemented with protease inhibitor cocktail for 30 min on ice with vortexing at 10-min intervals. After centrifugation, the supernatant was collected at 18,000g for 30 min as the nuclear fraction. Proteins were normalized by Bradford protein assay and analyzed by SDS-PAGE and immunoblotting with indicated antibodies.

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