Figure 3. Effect of NR2E3 mutations on DNA binding and transactivation. A: EMSAs were performed using the [³²P]-labeled Kni x2 probe with untransfected (UnTR) COS-1 cells or WT NR2E3 expressing cell extracts. Specificity of DNA binding is demonstrated by competition with unlabeled Kni x2 oligonucleotide (50×) and nonspecific (NS) oligonucleotide (50×). The arrow indicates the position of a specific DNA–protein complex between NR2E3 and Kni x2 oligonucleotide. B: Binding of mutant NR2E3 proteins to the labeled Kni x2 oligonucleotide was examined by EMSA. Mutant NR2E3 protein amount in cell extracts was normalized to WT-NR2E3 by immunoblot analysis. C: NR2E3 expression constructs were cotransfected into HEK293 cells with bovine rhodopsin-130 luciferase reporter plasmid and with NRL and CRX expression constructs. Fold change is relative to the empty expression vector control. Error bars indicate standard error of mean (SE). D: Luciferase assays were performed after co-transfection of mutant NR2E3 construct with the NRL and/or CRX expression constructs. ANOVA with a post hoc test were performed on each sample compared to WT NR2E3. Significant differences of p<0.05, and p<0.01 are shown as * and **, respectively. Error bars correspond to SEM.