Isolation of cytoplasmic, nuclear, and chromatin fractions and Western blot analysis.

Cells were lysed in cytosol extraction buffer (Tris-HCl, 10 mM, pH 8; sucrose, 0.34 mM; CaCl₂, 3 mM; MgCl₂, 2 mM; EDTA, 0.1 mM; dithiothreitol [DTT], 1 mM; Nonidet P-40, 0.1%; protease inhibitor cocktail). After centrifugation, the supernatant was collected as the cytoplasmic fraction. The pellet was then dissolved in nuclear extraction buffer (HEPES, 20 mM, pH 7.9; EDTA, 3 mM; glycerol, 10%; potassium acetate, 10 mM; magnesium chloride, 1.5 mM; DTT, 1 mM; Nonidet P-40, 0.5%; protease inhibitor cocktail) and collected as the nuclear fraction. Finally, the pellet was dissolved in chromatin extraction buffer (HEPES, 150 mM; MgCl₂, 1.5 mM; potassium acetate, 150 mM; glycerol, 10%; protease inhibitor cocktail), 4 U nuclease (DNase and RNase) was added, and the mixture was incubated for 30 min at 37°C. After centrifugation, the supernatant was collected as the chromatin fraction. The whole-cell lysates were prepared with cold lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1 mM EDTA, and protease inhibitor cocktail buffer tablet (PI; Roche Diagnostics) and resolved by SDS-PAGE. The various primary Abs used were mouse monoclonal anti-APE1 (Novus), anti-FLAG (Sigma), anti-HSC70 (catalog number B6-sc7298; Santa Cruz Biotechnology), anti-α-tubulin (catalog number T6199; Sigma), anti-AcAPE1, and anti-mouse Sin3a Ab (catalog number sc994; Santa Cruz).