Fig. 3. Zen interacts with the C15 enhancer. (A) Five overlapping fragments comprising the 650 bp enhancer were labeled at one end, incubated with GST-Zen protein, and subjected to DNAse I footprint analysis. Gray boxes at either end represent vector sequences. Only fragments IV (B) and V (C) displayed footprints. The first lane in each gel represents the chemical degradation of the probe at G+As. The second and third lanes contain fragments that were incubated without or with 60 ng of GST-Zen protein extract (denoted by the red rectangle) prior to DNAse I digestion, respectively. The regions protected by Zen are depicted as red ovals. The nucleotide sequence of the protected regions is shown beside the G+A lanes with putative core binding sites highlighted in red. (D) Gel comparing GST-Zen binding to wild-type and mutated oligonucleotides. Arrow, bound probe; two arrows, free probe. (E) DNA sequence of wild-type and mutated oligonucleotides used for mobility shift assays and in vitro mutagenesis. Mutations in Z1^m, Z2^m and Z3^m are denoted in green.