Figure 5.

RARα and RXR interacts with the novel intronic RARE in gel retardation assays. Nuclear extracts from E2-treated MCF-7 cells were incubated with biotin labeled oligonucleotide probes representing a consensus RARE, the wild-type DR2-2 or the mutant variant DR2-2mut (DR2-2-Mut1-S in Supplementary Table S2). Samples were resolved on a 6% non-denaturing polyacrylamide gel in TBE, transferred to Hybond N+ membranes and then incubated with streptavidin, and biotin-labeled DNA probes were detected by chemiluminescence. The name of the probe used in each binding reaction is indicated on the top of each panel. All binding reactions were competed with 200-fold molar excess of the corresponding unlabeled probe. Arrows point out the nuclear receptor–DNA complexes, while arrowheads point out the super shift. The asterisk indicates a DR2-2 independent interaction with nuclear proteins. Labeled probes were incubated in the absence of nuclear extract (lanes 1, 6 and 11), in the presence of nuclear extract alone (lanes 2, 7 and 12), nuclear extract together with an excess of competing unlabeled probe (lanes 3, 8 and 13), in the presence of RARα antibodies (lanes 4, 9 and 14), or in the presence of RXR antibodies (lanes 5, 10 and 15). Experiments were repeated at least three times and a representative result is shown.