Analysis of a mutated VWF E4BP4 binding element and ChIP assays implicate E4BP4 in modulating VWF promoter activity. (A) Mean normalized luciferase expression of the mutated and wild-type VWF E4BP4 binding element relative to the full-length proximal VWF promoter in BAECs. The P value from a t test to assess for a significant difference between the mutated and the wild-type VWF E4BP4 binding motif is indicated on the graph. (B) ChIP analysis of E4BP4-precipitated (precip) chromatin. PCR efficiency is demonstrated by amplifying appropriate fragments from the nonimmunoprecipitated input chromatin. PCR-amplified fragments consist of nt +3 to +225 of the VWF promoter. A region of the β-actin promoter²⁵  was used as a negative control. Lanes 1 to 3, 4 to 6, 7 to 9, and 10 to 12 represent chromatin isolated from HUVECs, HepG2, and undifferentiated and differentiated Dami cells, respectively. Lanes 1, 4, 7, and 10 used E4BP4 antibody no. SC9550, and lanes 2, 5, 8, and 11 used E4BP4 antibody no. SC9549X. Lanes 3, 6, 9, and 12 were precipitated with no antibody. (C) Mean normalized luciferase expression in HepG2 of the mutated and wild-type VWF E4BP4 binding elements relative to the full-length proximal VWF promoter with calculated significant difference between the mutated and the wild-type VWF E4BP4 element. (D) Comparison of mean normalized luciferase expression of the full-length proximal promoter in HepG2 cells with and without the E4BP4 expression plasmid. (E) Comparison of mean normalized luciferase expression of the wild-type and mutant E4BP4 binding elements in HepG2 cells cotransfected with the E4BP4 expression plasmid. Error bars represent standard error of the mean.