FIG. 2.

USF1 and USF2 DNA-binding increases at E-box motifs in the Gata4 and Nr5a1 promoters in differentiating Sertoli cells. A–C) In EMSAs, radiolabeled probes containing E-box regions of the Gata4 promoter (A), the Nr5a1 promoter (B), or a consensus CRE-binding motif for the CREB transcription factor (CRE probe; C) were incubated with nuclear extracts of Sertoli cells prepared immediately after isolation from 5- and 11-day-old testis. Representative images of the resulting DNA-protein complexes are shown, and quantitation of the mean ± SEM of four independent experiments is provided. The relative binding to the Gata4 and Nr5a1 promoters for each condition was normalized to CREB binding for each experiment, and the value of binding activity from 5-day-old Sertoli cell extracts was set equal to one. The relative CREB-binding activity represents the mean ± SEM of the fold-inductions derived directly from the intensities of the protein-DNA complexes. Representative full-length gel images for A–C are provided in Supplemental Figure S2. *P < 0.05. D) DNA-protein complexes from nuclear extracts isolated from 5- and 11-day-old Sertoli cells were incubated immediately after isolation with nonimmune sera or antisera against USF1, USF2, E47, E2A, or MYC or with no antisera. Supershifted DNA-protein complexes containing USF1 or USF2 are indicated. Exposure times for all complexes are less than 48 h. Free probe was run off the gel.