Fig 5.

USF2 binds to the human PAI1 promoter at multiple sites. (A) Cloned human PAI1 promoter/Luc reporter, showing the locations of consensus USF/HIF common (CACGTG), HIF (ACGTG), or USF2 (CANNTG, where the NN nucleotides are CG or GC) binding sites. The previously reported HIF or USF binding sites are represented by squares with bold borders, and the additional potential HIF or USF binding sites are represented by white squares. (B) Relative luciferase activity of PAI1/Luc reporter in response to activation by indicated plasmids in nanograms. (C) Gel shift assay of USF2 binding to potential USF2 binding sites on the PAI1 promoter using 30-mer double-stranded oligonucleotides centered at the indicated number and nuclear extracts prepared from USF2-transfected HEK293T cells. A 200-fold excess of cold WT competitor oligonucleotide or cold USF2-binding-site-mutated oligonucleotide (Mut) was added in competition assays.