Fig. 5. Identification of USF-1 as PAI-1 E-box probe-binding factor. A ³²P-body labeled PAI-1 wild-type (WT) E-box deoxyoligonucleotide probe was generated by PCR. Gel-purified probe was incubated with nuclear extracts from serum-stimulated EC-1 cells prior to UV irradiation and treated with DNase-1; the complexes were then boiled in sample buffer and resolved on SDS/9% polyacrylamide slab gels (A). A single major band at 44-45 kDa was resolved in the lane containing probe crosslinked to EC-1 nuclear extract (Probe+Extract). This band was not detected upon electrophoresis of probe alone (Free Probe) or in reactions where the probe was DNase-digested prior to addition of nuclear extract and UV crosslinking (Probe Digest). One low molecular weight nonspecific (ns) band was evident with the latter control. PAI-1 E-box-binding proteins were isolated from the nuclear fraction of growing EC-1 cells by tethered deoxyoligonucleotide affinity chromatography (B). Bound proteins were eluted and separated by gel electrophoresis. Western blotting confirmed USF-1 as one PAI-1 E-box target sequence binding element (B, Column). Two USF-1 species, corresponding in mobility to USF-1 (44 kDa) and phospho-USF-1 (P-USF-1, 45 kDa), were resolved by western analysis of extracts derived from growing EC-1 cells (B, Growing Cells). The predominant form of USF-1 eluted from PAI-1 deoxyoligonucleotide affinity columns co-migrated with the slower mobility (i.e. phosphorylated) USF-1 species. Acid phosphatase treatment (Phos) of nuclear extracts from serum-stimulated cells prior to gel electrophoresis and western blotting significantly decreased the abundance of the anti-USF-1 immunoreactive 45 kDa (P-USF-1) band compared with non-phosphatase-treated (Control) extracts (C).