Presynaptic Rab2 depletion mediated by RNAi knockdown caused accumulation of presynaptic proteins in neuronal cell bodies. (A) Western blot analysis of wild-type and Rab2-RNAi knockdown brains expressing the RNAi specifically in motoneurons (ok6-Gal4 driver) probed against Rab2 (top) and α-tubulin (bottom) as loading control. (B) Confocal images of neuronal somata from control (driver control) and Rab2-RNAi knockdown brains immunostained for the AZ scaffold protein BRP (green). Neuronal cell bodies in the VNC cortex (dotted lines). White squares show zoom area. Scale bar, overview, 10 µm; zoom, 2 µm. (C and D) Quantifications of the representative images of A. (C) Number of BRP aggregates (control: 100.0 ± 8.785%, n = 3; Rab2-RNAi: 180.1 ± 24.43%, n = 3). (D) BRP sum intensity (control: 100.0 ± 18.38%, n = 3; Rab2-RNAi: 346.6 ± 38.85%, n = 3). (E) Confocal images of neuronal somata of control (driver control) and Rab2-RNAi knockdown brains immunostained for BRP (green) and VGlut (magenta). (F) Quantifications of number of VGlut aggregates (control: 100.0 ± 16.85%, n = 4; Rab2-RNAi: 156.9 ± 11.86%, n = 4) of the representative images of E. (G) Confocal images of neuronal somata of control (driver control) and Rab2-RNAi knockdown brains immunostained for BRP (green) and Syt-1 (magenta). (H) Quantifications of number of Syt-1 aggregates (control: 100.0 ± 33.50%, n = 3; Rab2-RNAi: 367.6 ± 80.73%, n = 3) of the representative images of G. Scale bar for E and G, 2 µm. All graphs show mean ± SEM. n represents single VNCs (C and D) or single NMJs (one or two NMJs/larvae) for F and H. Normality was tested with the D’Agostino and Pearson omnibus normality test. If normally distributed (or assumed to be normally distributed for n < 7), the unpaired t test was used (C, D, F, H). n represents one larval brain from one animal. *, P < 0.05; **, P < 0.01. ctrl, control.