Nb200 209

Cell lines were grown to 70 to 80% confluence and lysed in RIPA (10% glycerol, 50 mM Tris–HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% NP40, 0.2% sodium deoxycholate, aqueous) containing protease and phosphatase inhibitors (Thermo Fisher Scientific) and benzonase (Santa Cruz). Protein concentrations were determined using the bicinchoninic acid (BCA) Protein Assay Kit, equally loaded and separated by SDS-PAGE, transferred to a nitrocellulose membrane, blocked in 5% milk, and incubated with primary antibodies overnight at 4 °C. Washed membranes were incubated for 45 min at room temperature in secondary antibody solution (LI-COR IRDye 680, 800; 1:10,000 in 5% milk), imaged on an Odyssey CLx, and analyzed using Image Studio Software (https://www.licor.com/bio/image-studio/). Antibodies were used at the following dilutions: β-actin (MilliporeSigma #A5316, 1:5000), HMOX1 (Abcam #ab13243, 1:1000), NRF2 (Cell Signaling Technology #20733, 1:1000), and NQO1 (Novus #NB200-209, 1:1000).

HepG2 cells were plated on 6-cm dishes at a density of ~500,000 cells/dish. The cells were transfected with NQO1 or negative control siRNA at 24 h after plating. After an additional 36 h of incubation, the cells were collected and lysed. Another set of cells plated in parallel were treated with dicoumarol or its solvent as a negative control at 48 h after plating and lysed after an additional 12 h of incubation. Whole-cell lysates were analyzed by western blotting using antibodies against alpha-tubulin (ab7291; Abcam), NQO1 (NB200-209; Novus Biologicals), and ODC1 (GTX101521; GeneTex) at the concentrations of 1/10,000, 1/1666, and 1/1000, respectively. Normalized ODC1 band intensity was calculated by dividing the ODC1 band volume in each condition by the corresponding band volume of alpha-tubulin. The experiment was performed in triplicate and repeated twice with equivalent results.