FIG. 1.

Schematic representation of the NR2E3 time-resolved fluorescence energy transfer (TR-FRET) assay. Unliganded apo-NR2E3 constitutively interacts with corepressor RetCOR, leading to association of 2 fluorescent probes, Eu cryptate and XL665, which are indirectly linked to GST-tagged NR2E3 and biotinylated RetCOR through Eu(K)-labeled anti-GST antibody and XL665-labeled streptavidin as shown in the picture. Light at 337 nm is used to excite Eu(K), which will emit at 620 nm. The proximity of the donor europium to the acceptor XL665 results in a transfer of energy between Eu(K) and XL665, which induces the specific FRET signal at 668 nm. Addition of an agonistic compound would prevent NR2E3–RetCOR interaction resulting in a signal decrease for TR-FRET at 668 nm.