Figure 4.

Subcellular localization of tagged Nrg180 constructs and in vivo effects of Nrg180 alterations on Myc expression, sensitivity to oxidative stress, and life span. A (left panel), Western blotting heads of controls (nSyb-Gal4) and animals expressing Nrg^HA (nSyb-Gal4,UAS-Nrg^HA). Csp served as a loading control. Right panels, Western blots of the membrane (5 heads) and soluble nuclear (80 heads) fractions (Subcellular Protein Fractionation Kit) of flies panneuronally (nSyb-Gal4) expressing Nrg^HA or Nrg^ICD-HA probed with anti-HA antibodies. Csp and lamin labeling served as fraction-specific loading controls. B, Western blots of the cytoplasmic (5 heads) and nuclear (80 heads) fractions (NE-PER® kit) of flies panneuronally (nSyb-Gal4) expressing Nrg^GFP probed with anti-GFP antibodies. Csp and lamin labeling served as fraction-specific loading controls. C, images of single confocal slices through the nucleus of the giant fiber soma of flies expressing tagged Nrg^HA, Nrg^ICD-HA, or Nrg^GFP (green) with the R68A06-Gal4 line. GF nucleus (magenta) was labeled by expression of UAS-mCherry-NLS. Scale bars, 5 μm. D, quantitative real-time PCR data showing the mean -fold expression of Drosophila Myc normalized to GAPDH in flies panneuronally overexpressing Nrg180 constructs (left) and in Nrg180 pacman mutants (right). Each sample was run in triplicate simultaneously with a nontemplate control. Error bars, S.E. of multiple independent experiments. *, p ≤ 0.05; **, p < 0.01; ***, p < 0.001 compared with nrg¹⁴;Pac[WT] using multiple-comparison one-way ANOVA. E, percent survival of animals panneuronally overexpressing Nrg^HA, Nrg^ICD-HA, Nrg^GFP, or untagged Nrg normalized to control flies (nSyb-Gal4/+). Male flies (3–5 days old) were kept in vials containing 20 mm paraquat, and survival was assessed after 72 h. The mean of 10 vials (20 flies/vial) was calculated for each genotype. The difference in the mean of each genotype was compared with nSyb-Gal4/+ control flies using multiple-comparison ANOVA (*, p ≤ 0.05). F, Kaplan–Meier survival analysis of animals panneuronally (nSyb-Gal4) overexpressing Nrg^HA, Nrg^ICD-HA, Nrg^GFP, or untagged Nrg180. Animals overexpressing Nrg180 (p = 0.0002), Nrg^HA (p = 0.002), and Nrg^ICD-HA (p = 0.01) were statistically significant (Holm–Sidak method) from WT controls (w¹¹¹⁸) and Nrg^GFP gain-of-function animals.