TRIM9 regulates SNARE complex formation and exocytosis. (A) Immunoblot showing SDS-resistant SNARE complexes in TRIM9^+/+ and TRIM9^−/− cortical neurons probed for VAMP2, Syntaxin-1a (Synt-1), and SNAP25. βIII-Tubulin (Tub) is a loading control. Syntaxin-1 and SNAP25 monomer are shown in boiled samples; n = 3 independent experiments. These samples are shown again in Fig. S4 A alongside boiled samples. (B) Quantitation of SNARE complexes (>40 kD) was normalized to βIII-tubulin. Significance is from ANOVA with Tukey’s post-hoc analysis; *, P < 0.5. (C) Inverted image montage of VAMP2-pHluorin exocytosis events in TIRF images. Arrowheads denote exocytic events, and time is noted in seconds. (D) Maximal projections of VAMP2-phluorin time lapse. Ovals denote location of exocytic events within 90 s (Videos 2 and 3). (E) Mean frequency of VAMP2-pHluorin exocytosis events ± SEM; n ≥ 10 cells/condition. P-value was obtained from Kruskal–Wallis nonparametric ANOVA within the entire image (top), soma (middle), or neurites (bottom). (F) DIC image montage showing the initial plasma membrane protrusion and subsequent axon branch formation of a TRIM9^+/+ cortical neuron after Netrin-1 stimulation (time is in hours and minutes). White arrowheads denote active plasma membrane protrusions; black arrowheads denote axon branches ≥20 µm long (Video 4). Scatter plot shows the time of initial axon protrusions at future branch sites in hours after Netrin-1 stimulation plotted against the time a stable 20-µm-long branch appears after Netrin-1 stimulation; n = 13 cells. Error bars show SEMs.