Fig. 7. Identification of Pax2-binding sites on the 2.4 kb GDNF promoter region. (A) Electrophoretic mobility shift experiments using isolated fragments or pools of fragments corresponding to the position of the published sequence as indicated. Increasing amounts (0, 1, 5 µl) of diluted Pax2-PD protein were used. The arrows indicate the shifted species in the fragment -237/-38 (PBS1) and +697/+968 (PBS2). PBS2 is also seen in the pooled fragments +500/+1054 (arrow). (B) Electrophoretic mobility shift experiments using increasing amounts of Pax2 protein and oligonucleotides corresponding to the predicted binding sites, based on DNAseI footprinting experiments. (C) Competition experiment using oligonucleotides for PBS1 and PBS2 and increasing amounts of excess (50x, 500x) unlabeled competitor oligonucleotide. (D) DNAseI footprint of PBS2 using increasing amounts of Pax2-PD bound to the fragment corresponding to the region of +697 to +968. Unbroken black bar indicates position of major protected region, with broken line indicating the extended footprint observed at higher concentrations of Pax2-PD.
