FIGURE 5.

EST is a transcriptional target of Nrf2. A, two AREs were predicted in the mouse EST gene promoter. The sequences of the two WT and mutant AREs are shown in boldface with the mutated nucleotides underlined. The ARE from the Nqo1 gene promoter is also shown. B, the binding of two ³²P-labeled putative AREs to Nrf2 homodimers or Nrf2-MafG heterodimers was confirmed by EMSA using in vitro-synthesized Nrf2 and MafG proteins. mt, mutant. C, ChIP assay to show the recruitment of Nrf2 protein onto the EST gene promoter. Primary hepatocytes were treated with vehicle or H₂O₂ (500 μm) for 12 h before ChIP using an anti-Nrf2 antibody. ChIP on Nqo1 was included as a positive control. The input contains the identical amount of DNA template that was used for the IgG or anti-Nrf2 antibody immunoprecipitation. Veh, vehicle. D, co-transfection of Nrf2 and MafG activated 3.0-kb but not 2.5-kb EST promoter reporter gene. Transfected cells were cultured for 24 h before the luciferase assay. *, p < 0.05 compared with the vector control.