FIGURE 2. TRR interacts genetically and associates in vitro and in vivo with components of the ecdysone receptor complex.

a, TRR NR2 binds EcR and USP. Bacterial extracts containing GST, GST–EcR or GST–USP were passed through a column containing the HA-tagged NR2 domain of TRR (Fig. 1a). Bound material was eluted and resolved by SDS–PAGE. b, Association of TRR with EcR and USP in vivo. Embryonic nuclear extracts were subjected to immunoprecipitation (IP) with an unrelated anti-GST, anti-HA or anti--Gal antibody, or with anti-TRR and anti-EcR antibodies. Immunoprecipitates were analysed by western blotting with the antibodies indicated on the right. c, Top, immunostaining of salivary gland polytene chromosomes with antibodies against the EcR-B1 isoform (green) and TRR (red), followed by Hoechst staining for DNA (blue). Bottom, high-magnification images of part of a chromosome. d, Left and middle, double immunostaining of an eye imaginal disc for TRR (red) and USP (green); right, immunostaining of a similar disc for EcR-A. Arrows indicate the morphogenetic furrow. Note that most of the region anterior to the morphogenetic furrow (left) is not stained with any of the antibodies. e, Clonal analysis of trr. Defects in eye structure in trr^1/trr¹ clones are marked by w⁺ (red); blue line on the left indicates a defect due to incomplete morphogenetic furrow progression; blue arrows indicate defects in ommatidial organization. f, Interaction of trr and Smr with a dominant-negative EcR mutant in the post-furrow retinal epithelium. Representative eyes were photographed through a dissecting microscope. GMR, GMR driver alone. F645A, GMR driver and EcR-F645A responder; P[trr]/F645A, GMR driver and EcR-F645A responder plus extra copy of trr; Smr^BG01648/F645A, GMR driver and EcR-F645A responder, hemizygous for a weak mutation in Smr (see also Supplementary Fig. 2b). g, Suppression of the B¹/trr¹ phenotype by EcR^M554. Double-headed arrow (same length in each panel) illustrates differing extents of the defect in morphogenetic furrow progression in different mutant combinations. h, Complete rescue of the trr⁴ mutant phenotype by EcR⁰⁶²¹⁰. In trr⁴/Y, the bracket indicates defects caused by incomplete morphogenetic furrow progression; the oval encloses defects in ommatidial structure.