Fig. 6. Quantitative analysis of the transcriptional activity of the Lef/Tcf binding sites in the Ep and EpExt enhancers. (A) NIH3T3 cells were transfected with the reporter constructs indicated and described in Fig. 6B. TK, TK minimal promoter. (B) The deletions of the EpExt enhancer analyzed by luciferase assay. Tcf/Lef binding sites (red circles) and a Gli site (green rectangle) are indicated; numbers indicate nucleotide position in the genomic sequence, as in Fig. 5A. (C-F) NIH3T3 cells were transfected with either the Ep (C,E) or the EpExt (D,F) enhancer driving firefly luciferase and the expression vectors indicated below each bar on the graph. Dp, stabilized ß-catenin; C, dominant-negative ß-catenin; Lef1N, dominant-negative Lef1. The values of fold induction represent the ratio of the firefly luciferase activity of cells transfected with and without the ß-catenin and Lef1 expression vectors, normalized to the activity of a control renilla luciferase-expressing vector. Independent experiments (n=10) were performed in triplicate and averaged.