2.8. Nuclear fractionation for chromatin‐bound and soluble protein fractions

C1 cells with Dox‐inducible shHMGA2 were treated with AZD2281 (olaparib) for 4 h prior to exposure to the alkylating drug MMS for 20 min. Cells were harvested immediately after MMS treatment for protein fractionation into chromatin‐bound and soluble nuclear proteins as described previously (Robu et al., 2013). Briefly, cells were resuspended for 5 min in whole‐cell lysis buffer containing 10 mm Hepes (pH 7.8) 0.34 sucrose, 10% glycerol, 10 mm KCl, 1.5 mm MgCl2, 1 mm PMSF, 0.1% Triton X‐100, and protease and phosphatase inhibitors. The cytoplasmic fraction was separated from nuclear pellet by centrifugation for 5 min at 4 °C and 1500 g. The nuclear pellet was lysed for 30 min on ice in nuclear lysis buffer containing 50 mm Tris/HCl (pH 7.8), 420 NaCl, 0.34M sucrose, 0.5% IGEPAL, and protease and phosphatase inhibitors. The chromatin fraction was separated from the nucleoplasm by centrifugation for 30 min at 16 000 g, and the pellet was suspended in chromatin lysis buffer containing 20 mm Tris/HCl (pH 7.5), 100 mm KCl, 2 mm MgCl2, 1 mm CaCl2, 0.3M sucrose, 0.1% Triton X‐100, protease inhibitors, phosphatase inhibitors, and 1 mm PMSF and briefly sonicated on ice‐water. The chromatin‐bound proteins were extracted by incubation with micrococcal nuclease (50 U·mL⁻¹) for 40 min at RT.