Fig. 4. Binding of a PAI-1 E-box probe by nuclear proteins from growing and wound-stimulated RK cells. The 18 bp ³²P-end labeled PAI-1 E-box probe (see Materials and Methods) was incubated with 10 µg of the nuclear protein fraction isolated from growing (A) or quiescent as well as wound-edge (B) RK cells and complexes resolved by gel electrophoresis. Two closely-spaced, `dumbell-shaped', bands (arrows) were evident in shifts produced by nuclear extracts derived from growing cells in the absence (none) of competing sequences (A). Incubation with the unlabeled WT PAI-1 18 bp E-box deoxyoligonucleotide (self) or the unlabeled standard consensus (SC) E-box construct (i.e. a CACGTG motif flanked by non-PAI-1 sequences) (both at a 50- to 100-fold molar excess) effectively blocked complex formation with the labeled probe. The AP-1 deoxyoligonucleotide and a mutant E-box construct (5'-CACGGA-3'), the latter in the context of PAI-1 flanking sequences, each failed to compete for probe binding (A). In contrast to PAI-1 probe patterns developed with nuclear extracts from quiescent (Q) RK cultures and which failed to form complexes that co-migrated with the slower mobile (i.e. upper) band, extracts prepared from wound-edge keratinocytes 2 hours post-scrape injury (W) produced the characteristic two-band complex (B).