Fig. 5. (A) Rig protein binds to DHR3, EcR, ßFTZ-F1, USP and SVP in vitro. In vitro synthesized ³⁵S-labelled Rig was incubated in a batch assay with control GST alone (lane 2) or GST fusions to the indicated receptors in either the presence (+) or absence (–) of ligand (1 µM muristerone A, a potent activator of EcR). Bound Rig was resolved by SDS-PAGE and visualized by autoradiography. Lane 1 depicts 1/5 the amount of input Rig protein, marked by an arrow. (B) Rig binds in vitro to the FTZ-F1 ligand binding domain (LBD) in an AF-2-independent manner. In vitro synthesized ³⁵S-labelled Rig was incubated in a batch assay with control GST alone (lane 2) or GST fusions to full-length FTZ-F1 (lane 3), full-length ßFTZ-F1 (lane 4), the ßFTZ-F1 LBD (E region, lane 5) or a mutated ßFTZ-F1 LBD in which the core of the AF-2 activation domain was deleted (AF-2, lane 6). Bound Rig was resolved by SDS-PAGE and visualized by autoradiography. Lane 1 depicts 1/5 the amount of input Rig protein, marked by an arrow.