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After dehydration, tongues were dried to the critical point using CO2 (Denton Vacuum DCP 1, Moorestown, NJ).
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Song, Z., McCall, K. & Steller, H. DCP-1, a Drosophila cell death protease essential for development.
Dcp-1 is a commonly used marker for cells undergoing apoptosis in Drosophila24.
To investigate whether the manipulation of autophagy using these two RNAi constructs would cause any ectopic apoptosis, we immunostained 10-day-old fly brains for Annexin V and activated Death caspase-1 (Dcp-1), respectively, to detect apoptotic cells.
We then incubated recombinant bacterially-expressed bicaudal with DCP-1 and analysed the resulting cleavage products by mass spectrometry.
We have cloned two of these substrates and confirm that they are efficiently cleaved by the Drosophila caspases DrICE and DCP-1.
As Fig. 3 E illustrates, only one of these mutants, D141A, fully resisted proteolysis by recombinant DrICE and similar results were found using DCP-1 (data not shown).
Upon activation within the apoptosome, Dronc can promote activation of other caspases such as DrICE and DCP-1 [23], [23], thereby initiating a protease cascade.
Dcp-1 and Dcp-1 were kindly provided by Kim McCall (Boston University).
ΔN-dcp-1 lacks the N-terminal prodomain of DCP-1.
We tested two additional Dcp-1 (Dcp-1 2 and Dcp-1 3 ) (Etchegaray et al., 2012) mutant lines that displayed the same phenotype as the RNAi result.
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