FIGURE 5.

Dorsal binding to the κB^P motif is co-regulator-dependent. A, EMSA was performed with the κB^I and κB^P motifs as probes. κB^P (AGAAAAACA) retards a larger complex (1st lane) compared with the κB^I (GGGAATTCC) (lane 3). 2nd lane (mut) shows competition with mutant oligonucleotide (mut-AGAATAATCC) where no Dorsal complex is retarded. B, diagrammatic representation of clustered AP1-binding sites in the dl promoter upstream of κB^P. Restriction enzymes used to delete the AP1 cluster are also shown. C, schematic representation of different reporter plasmids used for luciferase assay in experiments as explained in D and E. D, deletion of AP1 cluster in the dl promoter leads to constitutive expression of luciferase compared with control full-length reporter plasmid indicating the role of AP1 in dl repression. E, mutation in the κB^P motif also leads to constitutive expression of luciferase suggesting that Dorsal binding to the κB^P is also critical for dl repression. However, when the κB^I was mutated, no induction of luciferase was seen suggesting that the κB^I was required only for transcriptional activation of dl. The κB^P-κB^I promoter construct was used as the control plasmid.