FIGURE 3.
Mutations of the three NLS motifs in Nrf2 impair Nrf2-induced reporter gene promoter activity. In A, K562 cells were co-transfected with 0.2 μg of Gαi2 gene promoter construct (pGαi2(–1214/+115)-luc) and wild-type or mutated Nrf2 (0.3 μg) or the empty vector (pCI-Neo) followed by the addition of tBHQ (20 μm) 1 h later. In B, K562 cells were co-transfected with hNQO1-ARE-luc reporter gene construct (0.3 μg) and wild-type or mutated Nrf2 (0.2 μg) or the empty vector (pCI-Neo) followed by the addition of tBHQ (20 μm) 1 h later. For both panels, the cells were harvested for luciferase assay 20 h later as described under “Experimental Procedures.” The plotted values are means ± S.E. of duplicate assays for five experiments. Vector, empty vector (pCI-Neo); WT, plasmid containing cDNA for Nrf2 (pCI-Nrf2); Mt1, Mt2, and Mt3, pCI-Nrf2 mutated at NLS1, NLS2, and NLS3, respectively, as indicated in legend to Fig. 1A; DM 1,3, double mutant containing mutations at NLS1 and NLS3 as indicated in Fig. 1A.