The weak activator Nrf1β/LCR-F1 is negatively regulated by its CTD.

(A) The middle schematic representation of Nrf1β, Nrf1γ and their deletion mutants lacking various lengths of aa 297–741 of Nrf1 (a3). The contributions of the deleted regions to changes in the activity of Nrf1β and Nrf1γ, when compared with the background value, were examined using the P_SV40Nqo1-ARE-Luc reporter assay as described above. The right panel shows ARE-driven luciferase activity (a4) that was measured from COS-1 cells that had been co-transfected with each of numbered expression constructs and reporter plasmids. The data are shown as a fold change (mean ± S.D), and significant increases ($, p<0.05 and $$, p<0.001, n = 9) and decreases (*p<0.05, **p<0.001, n = 9) are indicated, relatively to the background value from transfection with an empty pcDNA3 control vector alone (C). The left two panels show western blotting of some of the above-transfected cell lysates with antibodies against either V5 (a1) or Xpress (a2). In addition, a non-specific protein-band is indicated (by arrow). The amount of protein applied to each polyacrylamide gel sample well was adjusted to ensure equal loading of β-gal activity. (B) COS-1 cells were co-transfected with each of the above-numbered expression constructs for Nrf1β, Nrf1γ and their mutants, together with an expression vector for wild-type Nrf1 (N1) or Nrf2 (N2), P_SV40Nqo1-ARE-Luc and β-gal plasmids. The cells were allowed to recover from transfection for 24 h before luciferase activity was measured. The data are shown as a fold change (mean ± S.D) of ARE-driven gene activity when compared with the background (value of 1.0). Significant increases ($, p<0.05 and $$, p<0.001, n = 9) and decreases (*p<0.05, **p<0.001, n = 9) are indicated. (C) The above-prepared cell lysates (b1 and b2) were resolved using 4–12% LDS/NuPAGE and visualized by western blotting with V5 antibody (c1 and c2). The electrophoresis band representing Nrf2 is indicated ((by arrow). The amount of protein loaded to each electrophoretic well was adjusted to ensure equal loading of β-gal activity.