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Fig. 4. Ey and So bind specifically to the core eye-enhancer.(A) Sequence of two potential binding sites for So (red) and Pax6 (blue) present in probe III. Consensus sequences are shown above. The sequence changes introduced to disrupt protein-DNA interactions are underlined. (B) Schematic showing the six overlapping probes (I-VI) used in EMSA. Green bars mark the conserved sequences A1 and A2. The relative position of the So and Pax6 sites within A1 is indicated. (C) EMSA with Ey protein. The position of the shifted Ey-III complex is marked by an arrow. Ey shifts probe III (lane 4) but not other probes (lanes 2,3,5,6,7). The binding of Ey to probe III can be competed by unlabeled III DNA (lane 10) but not by unlabeled Pax6^MUT-III DNA (lane 11). Probes (I-VI), protein (Ey) and/or competitor DNA (S,NS) added to each reaction are listed above each lane. S, specific: non-labeled probe III DNA. NS, non-specific: non-labeled probe III with the Pax6^MUT site. Reticulocyte lysate (L) only was added in the negative controls (lanes 1,8). The lower band (arrowhead) reflects a non-specific shift due to the lysate. (D) EMSA with So protein (GSTSo^SixHD). The position of the shifted GSTSo^SixHD-III complex is marked by an arrow. GSTSo^SixHD shifts probe III (lane 4) but not the other probes (lanes 2,3,5,6,7). The binding of So to probe III can be competed by unlabeled DNA with a So-binding site, but not by unlabeled So^MUT-III DNA. Probes (I-VI), protein (GSTSo^SixHD) and/or competitor DNA (S,NS1,NS2) added to each reaction are listed above each lane. GST was added to the negative controls (lanes 1,8). S, unlabeled so^AE oligo [So-binding site from Pauli et al. (Pauli et al., 2005)]; NS1, unlabeled probe II DNA; NS2, unlabeled probe III with the So^MUT site.

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