Figure 5. Specific nuclear protein binds to OTX2 binding elements in the human tyrosinase promoter. ARPE-19 cells were cultured for 14 days, and nuclear extracts were prepared. Binding of nuclear proteins to labeled OTX2 probes 1 and 2 (A) and 3 (B) was competed with only a sixfold excess of unlabeled probes for unrelated GAL4 (lanes 3), wild-type or mutated OTX2 sites 1 and 2 (lanes 4–7), wild-type or mutated OTX2 site 3 (lanes 8–9), and SNP rs4547091 at OTX2 site 3 (lanes 10). Complexes showing specific DNA-nuclear protein interaction are indicated with the symbol A, A1, A2, A3, or C. Non-specific DNA-nuclear protein complexes are indicated with B. Free probes in the absence of nuclear extracts are on lanes 1, and full reactions without competitor are on lanes 2. Above the gel images, intensities of the most specific DNA nuclear protein complexes A (A) and A1 (B) are shown relative to nuclear extracts without competitor (lanes 2=100). C: Supershift assay of OTX2 binding site 3 was performed by incubating nuclear extracts from ARPE-19 cells with or without the OTX2 antibody. Strong and modest inhibition of the specific complexes C1 and C2, respectively, was observed. D: D407 cells were transfected with the Flag-tagged OTX2 cDNA or with empty control plasmid (pCR3), and nuclear extracts were prepared and incubated with the Flag-specific antibody. The supershifted DNA–protein complex is indicated with the symbol S. Both supershift assays were controlled by using a non-specific control antibody (data not shown).