Figure 6. Attachment of the NTD from Nrf1 to Nrf2 results in negative regulation of the resulting fusion protein and their targeting to the endoplasmic reticulum.

(A) Keap1^−/− MEFs were co-transfected with 2 μg of a pcDNA3.1/V5His B expression construct for either Nrf1, Nrf2, NTD–Nrf2^f or NTD–Nrf2^s, together with 1 μg of pDsRed2-ER. Confocal imaging was performed as described in the legends to Figures 3 and 5. The bar represents 20 μm. (B) Shows keap1^+/+ and keap1^−/− MEFs that were co-transfected with 2.6 μg of an expression construct for either Nrf2, NTD–Nrf2^f, NTD–Nrf2^s, or empty pcDNA3.1/V5His B, along with 1.2 μg of P_TKnqo1-ARE-Luc and 0.2 μg of β-gal reporter plasmids. (C) Shows keap1^−/− MEFs co-transfected with 3.8 μg of DNA comprising 1.3 μg of either Keap1 expression construct or empty pcDNA3.1/V5His B, together with 1.3 μg of an expression construct for either Nrf2, NTD–Nrf2^f, Nrf1^Δ2-120 or empty pcDNA3.1/V5His B, 1.0 μg of pGL-6×ARE-Luc and 0.2 μg of β-gal reporter plasmids. Luciferase activity was measured and normalized to β-gal activity, and is presented as a fold change (means±S.D.) for two independent experiments performed in triplicate. (D) Co-transfection into COS-1 cells with each of the indicated Gal4D–Nrf1 fusion constructs with pDsRed2-ER. Immunocytochemistry and confocal imaging were performed as described in the legend to Figure 3.