Site-directed mutagenesis of E-boxes in HepcP1.1-luc. All 4 E-boxes (Figure 2) were simultaneously mutated to produce HepcP1.1-lucMtE4. (A) Basal activities of both mutant and wild-type promoters were compared with the MannWhitney U test following transfection into BHK cells. ***P < .001. (B) Cotransfections of wild-type and mutant promoters with USF1/USF2 or (C) c-Myc/Max expression plasmids to determine transactivation levels. In panels B and C, ▦ and ▪ represent activation profiles for HepcP1.1-lucMtE4 and HepcP1.1-luc, respectively. Fold activation is a ratio of transactivation by USF1, USF2, c-Myc, and Max, and basal expression levels of the respective constructs. P values from pairwise comparison between transactivation of the wild-type or mutant promoters are indicated; these were determined by 1-way ANOVA/Student-Newman-Keuls test. Two independent experiments were performed; samples in each assay were run in duplicate. Data are presented as means ± SEM.