Figure 3. The ETGE and DIDLID/DLG motifs in Nrf1 neither regulate its activity nor control its subcellular localization.

(A) Diagrammatic representation of constructs expressing wild-type Nrf1 and deletion mutants lacking sequences around the Neh2L subdomain. (B) These Nrf1 expression constructs, together with P_TKnqo1-ARE-Luc or P_SV40nqo1-ARE-Luc, and β-gal reporters, were co-transfected into either COS-1 or RL-34 cells. At 36 h after transfection, luciferase-reporter-gene activity was measured. The results represent fold changes (means±S.D.) from the mean activity measured in three separate wells from three independent experiments. Ectopic expression of proteins in the above cell lysates was determined using LDS/7% NuPAGE® followed by immunoblotting with an anti-V5 antibody (lower panels). Samples loaded in each well contained equal amounts of β-gal activity. (C) Immuocytochemistry was performed in COS-1 cells that were transfected with 1.3 μg of expression constructs for wild-type Nrf1 or the mutants Nrf1^ΔETGE and Nrf1^ΔDIDLID/DLG, as well as Nrf2/pcDNA3.1/V5His B or empty vector. Confocal images were obtained using a LSM 510 laser scanning microscope. FITC-labelled secondary antibody was used to locate V5-tagged Nrf1 or Nrf2. The DAPI signal represents nuclear DNA. ‘DIC’ signifies images obtained by normal optical microscopy. The merge signal represents the results obtained when the three images were superimposed. The length of the horizontal bar is 20 μm. (D) The cytoplasmic–nuclear distribution of the FITC signals was quantified by calculating the percentage of cells (100 cells were counted) with predominantly cytoplasmic (C) and nuclear (N) staining.