Figure 2.

ZEB1 protein from cultured human corneal epithelial cell (HCEpC) extracts binds to the proximal and distal E2-boxes in the COL4A3 promoter region. EMSA was performed using cultured HCEpC extracts as a source of ZEB1 protein and oligonucleotide probes containing both the proximal (E2^prox) and distal (E2^dist) E2-box motifs upstream of COL4A3. Lane 1: no complex was formed with the addition of ³²P-labeled E2^dist and E2^prox probe only. Lane 2: protein(s) in the HCEpC extracts interacted with ³²P-labeled E2^dist and E2^prox probes, forming complexes of the same location and similar intensity. Lanes 3 and 4: the addition of unlabeled E2^prox and E2^dist probes in (lane 3) 50- and (lane 4) 100-fold excess compared with the labeled probe resulted in the disappearance of the complex observed in lane 2. Lane 5: the addition of ZEB1 antibody E20X to the HCEpC extracts and ³²P-labeled E2^dist and E2^prox probes resulted in replacement of the band seen in lane 2 by a slower migrating band. Lane 6: addition of an antibody to heat shock factor I (HSF1) to the HCEpC extracts and ³²P-labeled E2^dist and E2^prox probes did not alter the migration of the complex seen in lane 2. Lanes 7 and 8: the addition of mutant probes M5 (lane 7) and M6 (lane 8) in 100-fold excess compared with the ³²P-labeled E2^dist and E2^prox probes did not result in an alteration of the complex observed in lane 2.