2.2. Western Blot analysis and NQO1 Enzyme Activity Assays

NQO1+ and NQO1– 231 cells were lysed in ice-cold RIPA with protease and phosphatase inhibitors (Santa Cruz, Dallas, TX) and whole-cell extracts were prepared by centrifugation (14000 rpm, 15 min) to remove insoluble components. Protein concentrations were determined by using a BCA assay (Thermo Scientific, Waltham, MA) to normalize the loading volumes. Proteins were separated by a 4–20% gradient SDS-PAGE gel (Bio-Rad, Hercules, CA) and transferred onto PVDF membranes. Primary antibodies for protein detection included: NQO1 (monoclonal mouse, 3187S, Cell Signaling Technology) and α-tubulin (monoclonal mouse, DM1A, Sigma) and were performed in Sigma 1X casein blocking buffer at 4 °C overnight. Secondary HRP-conjugated antibodies were incubated for 1 h at room temperature, followed by detection with SuperSignal West Pico (Thermo Scientific). Bands were quantified by the mean intensity using NIH Image J and normalized to α-tubulin. NQO1 enzyme activities for the cell lines were measured as dicoumarol-inhibited units as described elsewhere (Pink et al. 2000).