E4BP4 binds to the VWF promoter element and represses transcription in HepG2 cells. EMSA and supershift analysis of nuclear proteins isolated from (A) HepG2 cells transfected with the E4BP4 expression plasmid and (B-E) nuclear extracts from non–E4BP4-transfected HepG2 cells. Oligonucleotide probes contained the E4BP4 consensus binding element (E4BP4) or the VWF promoter E4BP4 binding element (Neg 5) or (C) a mutated VWF E4BP4 binding element (Neg 5 Mut). To supershift the DNA/protein complexes, an E4BP4 antibody (Ab) was used. An * identifies the very faint supershifted complex. (D) Specificity of the supershifted complex was confirmed by replacing the E4BP4 antibody with preimmune serum. (E) Competition binding assays using ³²P-labeled oligonucleotides for either E4BP4 or Neg 5 and competed away with unlabeled E4BP4, Neg5, or Neg 5 Mut oligonucleotides. NS indicates the presence of a nonspecific DNA/protein complex, and the free probe and well origin are shown. (F) HepG2 cells cotransfected with increasing concentrations of the E4BP4 expression vector and the various reporter constructs containing the indicated oligonucleotides upstream of the SV40 promoter. The mean normalized luciferase activity is expressed as fold repression relative to the reporter construct in the absence of the E4BP4 expression vector. Error bars represent standard error of the mean. A t test assessed for significant repression between cotransfection of the reporter construct in the absence or presence of the highest levels of the E4BP4 expression construct. P values are indicated above the constructs tested.