Figure 4. Syn5 SNARE complex formation is not affected by Retro-2.

(a) Representative confocal acquisitions of the cellular distribution of Syn5, GS27, and GS28 (green) in control (0.05% DMSO) or 25 μM Retro-2-treated cells, from 2 independent experiments. Nuclei marked by DAPI (blue). Scale bar = 10 μm. (b) Quantification of Syn5, GS27, or GS28 immunolabeling in the Golgi area, as defined by TGN46. Graph represents mean +/- SD for 180 (- Syn5), 43 (+ Syn5), 109 (- GS27), 43 (+ GS27), 71 (-GS28), 108 (+ GS28), 180 (- TGN46), 182 (+ TGN46) cells from 2 independent experiments. Each point represents one cell. One-way ANOVA was performed overall (P < 0.0001) with Sidak’s multiple comparisons test comparing untreated and Retro-2 treated conditions for each protein; Syn5: *** P < 0.0001; GS27: NS P = 0.1405; GS28: NS P = 0.7373; TGN46: NS P = 0.3446. (c) Representative confocal acquisitions of proximity ligation assay (PLA) of Syn5 with either GS27 or GS28 on 0.05% DMSO (control) or 25 μM Retro-2-treated cells. Scale bars = 10 μm. Representative images from 2 independent experiments. (d) Quantification of fluorescent dots / μm² as shown in (c). Number of dots were normalized per μm². Graph shows mean +/- SD for 25 (- GS27), 30 (+ GS27), 31 (- GS28), 39 (+ GS28) cells from 2 independent experiments. Each point represents one cell. One-way ANOVA was performed overall (*** P < 0.0001) with Sidak’s multiple comparisons test comparing untreated and Retro-2 treated conditions for each protein; GS27: NS P = 0.5077; GS28: NS P = 0.9997. (e) Western blotting of a representative GFP-Syn5 pull-down from 2 independent experiments. GFP-transfected cells were used as controls. GFP-Syn5 cells were treated either with 0.05% DMSO (control), or with 25 μM Retro-2.1. Note that GS27 and GS28 were pulled-down with Syn5, irrespective of incubation with Retro-2.1. See Supplementary Fig. 7b for full gels.