Chromatin Fractionation and Immunoblot Analysis

Chromatin fractionation was performed as described [38 (link), 61 (link)]. The preparation was carried from duplicate samples to enable presentation of all fractions. Approximately 1 × 10⁷ cells were washed in PBS and resuspended in 200 μl of solution A (10 mM Hepes [pH 7.9], 10 mM KCl, 1.5 mM MgCl₂, 0.34 M sucrose, 10% glycerol, 1 mM DTT, and protease and phosphatase inhibitors). Triton X-100 was added to a final concentration of 0.1%, cells were incubated on ice for 5 min, and the cytoplasmic (S1) and nuclear fractions (P1) were harvested by centrifugation at 1,300 × g for 4 min. Isolated nuclei were then washed in solution A, lysed in 150 μl solution B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, and protease and phosphatase inhibitors), and incubated on ice for 10 min. The soluble nuclear (S2) and chromatin fractions (p2) were harvested by centrifugation at 1,700 × g for 4 min. Isolated chromatin was washed once with solution B and spun down at high speed (10,000 × g for 1 min). Finally, chromatin was resuspended in 150 μl of SDS sample buffer and sheared by sonication. Protein concentrations were determined by BCA assay (ThermoScientific, Rockford, IL). Equal amounts of protein from whole cell extracts or from the different cellular fractions were mixed with 5 × laemmli buffer (Bio-Rad) and analyzed by immunobloting.