Anti ogg1

Cells (3×10 5 per well in 6-well plates) were harvested, and the pellet re-suspended in the cytoplasm extract (CE) buffer (10 mM HEPES, 60 mM KCl, 1 mM EDTA, 0.075% (v/v) NP40, 1mM DTT and 1 mM PMSF, adjusted to pH 7.6). After 5 min incubation on ice, the pellet was centrifuged at 1500 rpms for 5 min, and the supernatant containing the cytosolic fraction obtained. The remaining pellet was washed in CE buffer, and lysed in the nuclear extract (NE) buffer (20 mM Tris-HCl, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM PMSF and 25% (v/v) glycerol, adjusted to pH 8.0). After 20 min incubation on ice, the pellet was centrifuged at 12000 rpms for 10 min at 4°C, and the supernatant was collected. The nuclear extracts were stored at -80°C until used. For western blot analysis, protein samples (50 µg per lane) were resolved using 4-12% SDS-PAGE (Life Technologies), and transferred to nitrocellulose membranes, and incubated overnight with anti-NRF2 (Cell Signaling Technology, Danvers, MA) and anti-OGG1 (Origene, Rockville, MD).
Lamin (Bethyl, Montgomery, TX, USA) was used as loading control. After incubation with an HRP-conjugated secondary IgG (Sigma), the blots were developed using the ECL detection system (Pierce Biotechnology, Rockford, IL, USA). Band intensities were visualized by ChemiDoc using the Quantity One software (BioRad, Hercules, CA).