FIG. 4.

Prox1 represses Ff1-mediated transactivation in zebra fish embryos. (A) Embryos at the one- to two-cell stage were injected with the pLuc3xFRE reporter and different combinations of Ff1 and Prox1 expression vectors. pRL-CMV was included as a normalization control for firefly luciferase activity. Embryos (n = 5) were harvested at 14 hpf, and dual-luciferase assays were performed. The normalized luciferase activity was calculated from three experiments. (B) Electrophoretic mobility shift assay for zebra fish Ff1 proteins and Prox1, using a consensus FRE. In vitro-translated zebra fish Ff1a, Ff1b, Ff1c, and Prox1 and mouse SF-1 were allowed to bind DIG-labeled oligonucleotides carrying a consensus FRE. All zebra fish Ff1 proteins and mouse SF-1 bound FRE, but no binding was observed for zebra fish Prox1. Unlabeled FRE oligonucleotides (25-fold excess) were used as competitors to examine the specificity of the protein-FRE binding. The FRE-Ff1 complexes are denoted by an asterisk.