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. 2021 Feb 18;9:637565. doi: 10.3389/fcell.2021.637565

FIGURE 5.

FIGURE 5

SNAP29 interacts primarily with STX18 and is required to stabilize interactions with SEC22B. (A) HeLa cells expressing GFP–SNAP29Q1Q2. Cells stained with anti-Golgin97 to mark the Golgi apparatus (GA) show that GFP–SNAP29Q1Q2 forms enlarged bodies at the cell periphery and that the GA is fragmented. (A’) Quantification of Golgin97-positive GA object upon expression of the indicated transgenes reveals that GFP–SNAP29Q1Q2 induces GA fragmentation, thereby acting as a dominant negative SNAP29 form. The mean with standard error of the mean is shown, and the p-value is obtained by one-way ANOVA with Tukey’s multiple-comparisons analysis. (B) Representative images of a CLEM analysis of a HeLa cell expressing GFP–SNAP29Q1Q2. Single sections of HeLa cells expressing GFP–SNAP29Q1Q2 collected at phase contrast (bright field) and by confocal microscopy (GFP) to visualize the cell morphology and GFP–positive bodies. (B’) Electron microscopy image of the cell indicated by the arrow in panel (B). The GFP–SNAP29Q1Q2 bodies are composed of vesicular material and fragmented GA cisternae as highlighted in a close-up of the cytoplasmic portion boxed in panel (B’). (C,D) Single sections of HeLa cells over-expressing the indicated SNAP29 forms stained as indicated and acquired by stimulated emission depletion microscopy. The dashed and the continuous lines delimit the nucleus and the plasma membrane, respectively. High magnifications of the boxed areas are shown in the insets. The GFP–SNAP29Q1Q2 bodies are highly decorated with N-ethylmaleimide-sensitive fusion. (E) Immunoblotting (IB) with the indicated antibodies of proteins immunoprecipitated using GFP Trap from protein extracts of HeLa cells expressing the indicated transgenes and related control. Interactions with Qb, Qc, and R-SNAREs, but not with Qa-SNARE STX18, are weakened by the expression of GFP–SNAP29Q1Q2. (F,G) IB with the indicated antibodies of protein extracts from HeLa cells over-expressing the indicated transgenes (F) or treated as indicated (G) and immunoprecipitated with anti-SEC22B and related controls. The asterisk indicates a non-specific band recognized by anti-SNAP29. GFP–SNAP29Q1Q2 is not included in SEC22B immunoprecipitations, and SNAP29 depletion impairs the interaction of SEC22B with STX18.