Promoter occupancy by c-Myc/Max and USF1/USF2 in vitro and in chromatin. (A) HepG2 cell nuclear extracts (10 μg) were incubated with radiolabeled E-boxes (including the mutant E-box, Emt); where indicated, excess unlabeled or cold competitor oligonucleotide (100 pmol) was added to the binding reactions. (B) Binding specificity was ascertained without or with USF1 and USF2 antibodies in a supershift assay. Arrows indicate E-box–nucleoprotein complexes; NS, nonspecific-binding nuclear components. **Supershifted complexes. (C) Binding of in vitro–translated USF1/USF2 and c-Myc/Max heterodimers to the E-boxes. L indicates E-box2 incubated with untranslated rabbit reticulocyte lysate as negative control; 1, E-box2 incubated with USF1/USF2 heterodimer; 2, E-box2 incubated with c-Myc/Max heterodimer; and 3, Emt incubated with c-Myc/Max heterodimer. Arrows show complexes of E-box and USF1/USF2 or c-Myc/Max heterodimers. (D) Arrangement of the 2 canonical E-boxes within ChIP target DNA; PCR primers (arrows) were chosen to amplify a 300-bp fragment of the promoter encompassing nucleotide sequences from –642 to –343. (E) ChIP assay. PCR was performed on whole chromatin without immunoprecipitation (Input) and on chromatin immunoprecipitated with either a nonspecific antibody (ns), or with (+) antibodies to c-Myc/Max together, USF1, and USF2. M indicates DNA molecular size marker. 2xE-box-luc was used as positive control (“C”) in PCR.