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. 2000 Aug 15;97(17):9443–9448. doi: 10.1073/pnas.97.17.9443

Figure 4.

Figure 4

Characterization of the transactivation domain of Nkx2.2. A reporter plasmid containing five tandem copies of the GAL4 UAS upstream of the E1b minimal promoter driving luciferase and an expression plasmid encoding each GAL4DBD-Nkx2.2 fusion construct were cotransfected with a CMV promoter-driven β-galactosidase expression plasmid. All luciferase activities are corrected for β-galactosidase activity. All data are shown as mean ± SEM. (A) The sequences of the native and mutant NK2-SD. The NK2-SD is underlined, and mutations are in boldface. (B) Western blotting data. Each construct is transfected into NIH 3T3 cells. Five micrograms of nuclear extracts from each cell is loaded. (C) The relative luciferase activities from βTC3 cells and NIH 3T3 cells transfected with expression plasmids encoding the C-terminal region with wild-type or mutant NK2-SD fused to the GAL4DBD. Relative luciferase activities are calculated, with the activity of cells transfected with the GAL4DBD alone set at 1. (D) The relative luciferase activities from βTC3 cells and NIH 3T3 cells transfected with expression plasmids encoding the full-length Nkx2.2 with wild-type or mutant NK2-SD fused to the GAL4DBD. Relative luciferase activities are calculated, with the activity of cells transfected with GAL4DBD–full-length Nkx2.2 alone set at 1. Comparison with the GAL4DBD alone is shown in Fig. 2B. (E) Relative luciferase activities from cells transfected with the VP16 activation domain or Pax6 activation domain fused to wild-type or mutant NK2-SD and the GAL4DBD. Relative luciferase activities are calculated, with the activity of cells transfected with an expression plasmid containing the isolated VP16 activation domain fused to the GAL4DBD set at 1. VP16 activation domain fused to the GAL4DBD showed 516,000-fold activation in NIH 3T3 cells and 5,990-fold activation in βTC3 cells compared with the GAL4DBD alone. On the other hand, Pax6 activation domain fused to the GAL4DBD showed 102-fold activation in NIH 3T3 cells and 6.1-fold activation in βTC3 cells compared with the GAL4DBD alone.