Anti nqo1 antibody

Cells were treated with 5 or 10μM SFN for 24 hours, lysed and fractionated to obtain cytoplasmic and nuclear fractions. The nuclear extract was used for Western blotting of NRF2, and the cytoplasmic extract for HO1 and NQO1. Western blotting was done with a standard protocol. In brief, 20μg of protein was resolved on a SDS-polyacrylamide gel and transferred to nitrocellulose membranes. NRF2, HO1, and NQO1 were detected with a rabbit polyclonal anti-NRF2 antibody (1:1000; Abcam), a rabbit polyclonal anti-HO1 antibody (1:2000; Abcam), and a mouse polyclonal anti-NQO1 antibody (1:1000; Cell Signaling, Danvers, MA), respectively. Membranes were developed with ECL chemiluminescence and exposed on X-ray film. Quantitation of the bands on the film was carried out by a densitometer. Mouse tongue samples were homogenized and lysed in RIPA buffer for Western blotting of NQO1, HO1 and GCLC in a similar way. GAPDH or β-actin was detected as a loading control.

Total cellular protein was extracted in RIPA buffer (Nacalai Tesque) containing a protease inhibitor cocktail (Nacalai Tesque). The protein concentration was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific). Ten micrograms of the total protein extract were subjected to SDS–PAGE. After electrophoresis, the proteins were transferred onto a PVDF membrane using the Trans-Blot Turbo transfer system (Bio-Rad). Immunoreaction was performed using Immobilon^® GO (Millipore, Billerica, MA, USA) according to the manufacturer’s protocol. Anti NQO1 antibody (Cell Signaling, Danvers, MA, USA) and HRP-conjugated anti-rabbit IgG were used in the immunoreaction. The protein bands were visualized using Western Lightning Clarity Western ECL Substrate (Bio-Rad). The intensity of each band was quantified using an image analyzer (LAS-2000, Fujifilm, Tokyo, Japan).