Figure 3. No evidence for a role of Drep-2 in regulation of apoptosis.
(A) Synaptosome-like preparation of adult wild-type head extracts (Depner et al., 2014), probed with Drep-2C-Term. Drep-2 is concentrated in fractions containing synaptic membranes. S = supernatant, P = pellet, L = (after) lysis. Please see the protocol by Depner et al. (2014) for a more detailed explanation of the fractionation procedure. (B) Mutants (drep-2ex13) did not show a rough eye phenotype. The facet eyes of flies, highly ordered structures, are often affected in apoptosis mutants. By contrast, the eyes of drep-2 mutants appeared normal. (C) The number of mb247-positive KCs does not differ between drep-2ex13 mutants and controls. GFP was expressed using the MB KC driver mb247-Gal4. GFP-positive cell bodies were counted and compared between genotypes. No significant difference was found between mean cell body counts (Mann–Whitney U test, p = 0.886). Average cell body counts were in the expected range: control = 651, mutant = 669, published = 700 (Schwaerzel et al., 2002). (D) Purified Drep-2 does not degrade linearized plasmid DNA. Left: SDS-PAGE of the final elusion profile of purified Drep-2, loaded onto a HighLoad Superdex S200 16/60 column. Right: Nuclease activity assay of purified Drep-2 analyzed by 1% (wt/vol) agarose gel. Drep-2 was incubated in a time course experiment with linearized plasmid DNA. No nuclease activity could be detected. Instead, Drep-2 seemed to precipitate DNA, as evidenced by high-molecular DNA not entering into the agarose gel when incubated with Drep-2 (arrow).