FIG. 7.

Functional analysis of a putative Sp1 binding site within the ORF 28/29 intergenic element. (A) Results of magnetic bead recruitment assays. Two hundred-fifty micrograms of uninfected MeWo cell nuclear extract was incubated with 10 pmol of immobilized 5′-biotinylated full-length wild-type (WT) or mutant (Sp1.1, Sp1.2, Sp1.3) intergenic regions. The sequences of the Sp1 binding site within the promoters are listed with the mutagenized nucleotides underlined and in bold. Following washing and elution, the binding of Sp1 and USF to the various DNA fragments were assessed by SDS-PAGE and immunoblotting of the eluates. (B) Schematic representation of the structure and target sequences of the WT and mutant dual-luciferase vectors containing mutagenized Sp1 (pRFL/Sp1m) and USF (pRFL/USFm) binding site used in transient transfection assays. (C) Results of transfection assays. Each pRFL vector (1.5 μg) was transfected into MeWo cells in the absence (solid bar) or presence of 0.05 μg of pCMV62 plasmid (open bar). The results are presented as described in the legend to Fig. 3C.