Figure 8.

DACH1 inhibits c-Jun transactivation, requiring the δ domain. (A) Schematic representation of c-Jun expression vectors. (B) The HEK 293T cells transiently transfected with expression vectors encoding either wild-type or mutant c-Jun as an E2 fusion proteins or a DACH1 mammalian expression vector. Western blot analysis is shown for antibodies to either c-Jun (E2) or DACH1 (Flag). GDI is used as a protein loading control. (C and D) HEK293T cells were transiently transfected with a luciferase reporter encoding a multimeric E2 binding site linked to a luciferase reporter [(E2)₆ LUC] and the mammalian expression vectors encoding either c-Jun-E2, Ha-Ras V12, or a DACH1 mammalian expression vector as indicated. Luciferase activity is shown as -fold change normalized to a β-galactosidase reporter gene used to normalize for transfection efficiency. Data are expressed as mean ± SEM for n = 7 separate transfections. (E) 3T3 cells were transfected with the vectors described in D, and the effect of shRNA to endogenous Dach1 was assessed. (F) Activity of an AP-1–responsive reporter gene was determined upon reduction of endogenous Dach1 through shRNA.