Quantification of Nrf2-ARE Binding Activity

Efficiency of Nrf2-ARE binding activity was measured using a commercial Trans-AM Nrf2 kit from Active motif. Briefly, wild-type or mutated competitor oligonucleotides bearing the antioxidant response element (ARE) consensus sequence were incubated with nuclear extract (10 μg) for Nrf2 and the bound Nrf2 was incubated with an anti-Nrf2 primary antibody (100 μl of a 1:1000 dilution) for 1 h. Further, HRP-conjugated secondary antibody (100 μl of a 1:1000 dilution) was added in each well and incubated for 1 h prior to chromogenic reaction with TMB substrate and the absorbance was measured at 450 nm with a reference wave length of 655 nm using a BioTek Epoch plate reader. Incubation with normal rabbit polyclonal IgG was also performed separately to confirm the specificity of the Nrf2 antibody (12 (link), 42 (link)).