Development and characterization of PRAP, a fluorescent probe for monitoring DIAP1 degradation. (A) Schematic representation of PRAP. (B) Cell death was not inhibited by PRAP expression in S2 cells. Cell viability (mean ± SD) was determined by a cell death assay 36 h after transfection with each indicator (1, 5, or 15 ng), 5 ng of pUAST-Reaper, and 10 ng of pWAGAL4 together with 100 ng of pCaSperR-hs-lacZ. (C–F) Eye phenotype of UAS-mycDIAP1/+; UAS-Reaper/+; GMR-GAL4/+ (C), UAS-Reaper/UAS-GFP; GMR-GAL4/+ (D), UAS-Reaper/UAS-DIAP1-Venus; GMR-GAL4/+ (E), and UAS-Reaper/UAS-PRAP; GMR-GAL4/+ (F). (G) PRAP is an unstable protein with a short half-life. S2 cells were treated with cycloheximide, and cell extracts were prepared at the times indicated. The expression levels of PRAP and the endogenous DIAP1 protein were detected in whole-cell lysates by immunoblotting with an anti-DIAP1 antibody. (H) PRAP degradation is mediated by the ubiquitin–proteasome pathway. 18 h after S2 cells were transfected with 20 ng of pUAST-PRAP and 5 ng of pWAGAL4, the cells were divided into two dishes and incubated for 5 h with or without lactacystin. The levels of PRAP and endogenous DIAP1 protein were detected by immunoblotting with an anti-DIAP1 antibody. (I–L) PRAP (green) is degraded before the nuclear condensation and morphological changes that accompany cell death. Arrowhead indicates a cell showing PRAP degradation. To mark each nucleus, the ECFP signal is shown in magenta. Bar, 20 µm. The genotype was en-GAL4 UAS-PRAP/+; UAS-Histone2B-ECFP/+.