Rpr self-association and its impact on apoptotic activity. (A) Secondary structure consensus prediction of the Drosophila Rpr. Nomenclature: c, disordered; e, β-strand; h, helical. Three distinct Rpr domains are distinguishable: the IBM motif (IBM_Rpr), a central helical domain (Helical Domain_Rpr), and a C-terminal unstructured tail (Tail_Rpr). The GH3 domain is marked with a red line above the amino acid sequence. Blue and red dots represent amino acids that were replaced by site-directed mutagenesis. Red dots represent amino acids that have an effect on the protein activities once replaced. (B) Pull-down (PD) experiment for testing the interaction between Rpr-GST and ³⁵S-Rpr (Rpr-GST PD). As a specificity control, GST (bait) was tested for interaction with ³⁵S-Rpr (GST PD). “Input” shows the expression of the radiolabeled Rpr and represents 10% of the protein amount used in the PD assay. “SDS-PAGE” indicates the amount of bait proteins used, as visualized by Coomassie staining. “Autoradiography” shows the radiolabeled proteins in the experiments. (C) Protein–protein interaction assay between Rpr-GST and ³⁵S-Rpr mutants. “SDS-PAGE” indicates the amount of Rpr-GST protein used as bait. “Input” lanes indicate the autoradiography detection of the in vitro–translated ³⁵S-Rpr mutants, for expression comparison. Each represents 10% of radiolabeled Rpr mutant amounts used in the PD assay. Rpr-GST PD is a pull-down assay, showing the binding of the individual ³⁵S-Rpr mutants to Rpr-GST. (D) Eye images of transgenic Drosophila expressing Rpr-HA and Rpr-HA mutants Q23ER26A and the GH3 mutant F34AL35A. Genotypes: ;GMR>Gal4/+;, ;UAS:Rpr-HA/GMR>Gal4;, ;UAS:Rpr-HA Q23ER26A/GMR>Gal4;, ;UAS:Rpr-HA F34AL35A/GMR>Gal4;.