2.10. PARP-1 Cleavage

Nuclear extracts were prepared by detaching the cells with trypsin-EDTA, incubating them on ice for 10 min in 10 mM Hepes, pH 8.0, containing 10 mM KCl, 1.5 mM MgCl₂, 0.5 mM DTT, and a complete EDTA-free protease inhibitor cocktail (Roche), lysing them by adding 0.1% NP-40, and centrifuging the lysates at 1,500 ×g for 10 min at 4°C. The pellet was dissolved in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing a protease inhibitor cocktail and incubated on ice for 30 min. After centrifugation at 1,500 ×g for 30 min at 4°C, the nuclear protein concentration was measured using the Bradford method [21], and 30 μg of the protein extracts was separated by 10% SDS-PAGE. Western blotting analysis was performed using the chemiluminescent method and the following antibodies: poly(ADP-ribose) polymerase-1 (PARP-1) mAb (C2-10, Santa Cruz, CA) diluted 1 : 2,000 in blocking buffer (5% BSA in TBS buffer containing 0.1% v/v Tween 20) or the B23 mAb (Sigma-Aldrich, St. Louis, USA; 1 : 2,000 dilution) as loading control for the nuclear proteins. An HRP-conjugated goat anti-mouse IgG was used for detection (Pierce, Rockford, IL, USA).