The cleaved-Caspase-3 antibody is a marker of Caspase-9-like DRONC activity in Drosophila

The cleaved-Caspase-3 antibody is a popular tool in apoptosis research in Drosophila. As the antibody was raised against cleaved human Caspase-3, it was assumed that it detects cleaved DRICE and DCP-1, Caspase-3-like effector caspases in Drosophila. However, as shown here, strong immunoreactivity persists in apoptotic models doubly mutant for drICE and dcp-1. In contrast, mutants of the apoptosome components DRONC (Caspase-9-like) and ARK (Apaf-1 related) do not label with the cleaved-Caspase-3 antibody. By peptide blocking experiments and further genetic studies, we provide evidence that the cleaved-Caspase-3 antibody recognizes multiple proteins including DCP-1 and likely DRICE, but also at least one additional unknown protein, all of which require DRONC for epitope exposure. The unknown substrate may be involved in non-apoptotic functions of DRONC. Because the cleaved-Caspase-3 antibody not only detects cleaved Caspase-3-like proteins in Drosophila, but also other proteins in a DRONC-dependent manner, it is more accurate to consider the cleaved-Caspase-3 antibody as a marker for DRONC activity, rather than effector caspase activity.