Functional analyses of the putative enhancer elements. (A) Luciferase fusions of E-box duplex oligonucleotides (only partial sequences are shown) with 1 or 2 E-boxes (details in “Materials and methods”). A single E-box (mhepcPE) conserved in both mouse hepcidin genes was similarly subcloned. (B) E-box transactivation assays. Constructs in panel A, 100 ng each, were transfected into BHK cells alone or with 100 ng each of USF1, USF2, c-Myc, and Max expression vectors and combinations thereof. Fold transactivation by USF1, USF2, c-Myc, and Max was expressed with respect to the basal activity of each E-box construct alone. Data represent means of 3 independent experiments (± SEM). *P < .05; **P < .005; ***P < .001; determined by 1-way ANOVA and the Student-Newman-Keuls comparisons test.