Figure 6. Deletion of Sp1_-520 site inhibits estrogen-mediated ERα promoter activity.

A. PCR products of the Sp1_-520 binding site after ChIP with ERα and ERβ antibodies, respectively, in uterine arteries. M indicates markers. B. A 2035 bp fragment of ovine ERα promoter region spanning −2000 to +35 bp relative to the transcription start site was amplified by PCR and inserted into pGL3 to yield the full-length promoter–reporter plasmid (WT). A site-specific deletion mutation at Sp1_-520 (ΔSp1) was constructed. Constructs were transiently transfected to uterine arterial smooth muscle cells. Cells were then treated with 10 nmol/L 17β-estradiol (E₂β) for 24 hours. Firefly and Renilla reniformis luciferase activities were measured in a luminometer using a dual-luciferase reporter assay system, and promoter activity was expressed as % wild type (%WT). Data are means ± SEM, * P < 0.05, versus WT; ⁺ P < 0.05, versus WT+ E₂β. n = 4