tBHQ induces NRF2 nuclear translocation and binding of NRF2 to the γ-globin promoter in K562 cells. (A) Simplified schematic of the NRF2/ARE pathway. Under basal conditions, NRF2 is sequestered in the cytoplasm by KEAP1, which is an E3 ligase substrate adaptor for the Cul3/Rbx1 complex that ubiquitinates NRF2, targeting it to the proteasome. Reactive oxygen species or electrophiles alter KEAP1 structure so that newly translated NRF2 is no longer ubiquinated and can accumulate and translocate to the nucleus where it heterodimerizes with small Maf proteins and binds the ARE in target gene promoters. (B) Western blot analysis of NRF2 protein in total cell and nuclear lysates after tBHQ treatment. (C) EMSA shows NRF2 binding to the γ-globin ARE region in vitro. The ARE region of the NQO1 promoter, and known GATA1 binding site are used as positive and negative controls for NRF2 binding. Unlabeled γ-globin promoter probe was used as in competition reactions. One microgram of IgG or NRF2 antibody was added and incubated for an additional hour after the binding reaction. The reactions were all run on the same gel. Different exposures are presented for clarity. (D) ChIP analysis of cells treated with tBHQ. Quantitative real-time PCR was performed on immunoprecipitated DNA with the use of primers that flank the ARE region in the γ-globin promoter. A total of 25μM tBHQ was used in these experiments. The NQO1 promoter was used as a positive control and necdin promoter was used as a negative control. *P < .05, **P < .01 compared with untreated control.