Materials and Methods.

Third instar larvae were dissected in PBS and fixed in 4% paraformaldehyde for 25 min at room temperature, then washed in PBST three times (each 10 min), blocked in blocking solution (PBST with 5% normal goat serum, 1% BSA) for 60 min. Primary antibodies were: guinea-pig anti-Nemy 1:20, rabbit anti-Nemy 1:20, mouse anti-Dlg 1:20 (DSHB), FITC-labeled goat anti-HRP1:250 (The Jackson Laboratory), rabbit anti-GFP 1:200 (Life Technologies). Secondary antibodies were: goat anti-guinea-pig Alexa-Fluor 488 1:300 (Life Technologies), goat anti-rabbit Alexa-Fluor 555 1:300 (Molecular Probes) or goat anti-rabbit Alexa-Flour 488 1:300 (Life Technologies), goat anti-mouse Alexa-Fluor 555 1:300 (Molecular Probes). All antibodies were incubated for 2 h at room temperature or overnight at 4°C. All images were obtained using a Leica DMRA2 fluorescent microscope (Leica, Deerfield, IL). S1 Fig A. Peptide amidation levels. (A1) Peptide amidation in control cantonised w1118 and Canton S lines. NS indicates non-significant difference. (A2) Peptide amidation in control Canton S and nemy mutant lines. * indicates a significant difference (P < 0.05) compared with w1118 and # indicates a significant difference (P < 0.05) compared with nemy26.2 mutants. B. Third instar larval NMJs. Muscles 6/7 were immunostained with guinea-pig #1 (B1)and guinea-pig #2 (B2) anti-Nemy. The arrows point to the center of synaptic boutons that express Nemy. C. Nemy is expressed at NMJs. Images of third instar NMJs formed on muscles 6 and 7 stained with rabbit anti-Nemy (C1), FITC conjugated goat anti-horse radish peroxidase (C2) and its co-localization (C3). The arrows point to the center of synaptic boutons that express Nemy. D. Nemy expression at NMJs can be detected using Nemy-GAL4. (D1) Double staining for nemy335GAL4:10XUAS-mCD8-GFP at the larvae NMJ, muscles 6/7. GFP fluorescence pattern, anti-DLG immunereactivity (D2) and merged red and green channels (D3).
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