Figure 8. Both AD2 and NST domains positively regulates chimaeric Gal4-Nrf1 and Gal4-Nrf1β factors.

(A) Schematic representation of expression constructs for Gal4D (Gal4 DNA-binding domain) fusion proteins containing various portions of Nrf1 or Nrf1β (left panel). They were created by ligation of their encoding cDNA fragments into the BamHI/EcoRI sites of the pcDNA3/Gal4-V5 vector. The left panel shows Gal4D-directed reporter activity that was measured from COS-1 cells had been cotransfected with each of indicated expression constructs for the various Gal4D/Nrf1 fusion proteins (1.2 µg), together with P_TKUAS×4-Luc (0.6 µg) and b-gal (0.2 µg) plasmids. The data are shown graphically as fold changes (mean ± S.D.) of transactivation by indicated Gal4-fusion factors 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) in activity relatively to the referenced activity are indicated (arrows). (B) The above-prepared cell lysates were resolved using 4–12% LDS/NuPAGE and examined by western blotting with V5 antibody. The electrophoretic bands representing free Gal4D and Gal4-Nrf1 fusion proteins are indicated. Samples loaded on each well were calculated to contain equal amounts of β-gal activity.