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Cell Death and Differentiation (2010) 17, 534539
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The cleaved-Caspase-3 antibody is a marker of Caspase-9-like DRONC activity in Drosophila
Y Fan1 and A Bergmann*,1
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.
Cell Death and Differentiation (2010) 17, 534539; doi:http://dx.doi.org/10.1038/cdd.2009.185
Web End =10.1038/cdd.2009.185 ; published online 4 December 2009
The cleaved-Caspase-3 (Asp175) antibody (referred to as cleaved-Caspase-3 antibody) from Cell Signaling Technology (Danvas, MA, USA) is a polyclonal antibody obtained from rabbit that was raised against a peptide in the large subunit of the human effector caspase, Caspase-3, amino-terminal to Asp175.1 The antibody does not detect unprocessed Caspase-3. However, after proteolytic cleavage between Asp175 and Ser176, separating the large and small subunits leads to the activation of Caspase-3, the epitope is exposed and can be detected by cleaved-Caspase-3 antibody, thus making the antibody a marker for cleaved and active Caspase-3 in dying cells. A similar antibody, termed CM1, has previously been described;2,3 however, the CM1 antibody is no longer available and is not a subject of this analysis.
Apoptosis in Drosophila is under the control of pro-apoptotic genes reaper, hid and grim.46 (reviewed in7) The products of these genes trigger apoptosis through the induction of proteolytic degradation of inhibitor of apoptosis proteins (IAPs), most notably DIAP1,811 to...