Fig 2: Fusion of cytotoxic granules with the plasma membrane is insensitive to Tetanus toxin. (A) Bead-stimulated human CD8+ T cells transfected with GFP or TeNT-GFP as indicated. VAMP2 and VAMP7 protein levels were determined by Western blot analysis 12–16 h after transfection. (B) Expression of VAMP2 and VAMP7 protein levels relative to GAPDH in CTLs transfected with GFP or TeNT-GFP as indicated. Graphs represent means [N = 3, ***p < 0.001 (Student's t-test)]. (C) Bead-stimulated human CTLs co-transfected with either GFP or TeNT-GFP along with granzyme B-mCherry and imaged 12 h after transfection. Representative live-cell TIRFM images of CTLs in contact with an anti-CD3 coated coverslip. Fusion events indicated with open arrowheads. (D) Mean percentage of cytotoxic granule fusion in cells transfected with either GFP (n = 50) or TeNT-GFP (n = 40), p = 0.704 (Student's t-test). (E) Mean average number of granules fused per cell over time in the TIRF plane in cells transfected with either GFP (n = 20) or TeNT-GFP (n = 15), p = 0.939 (Student's t-test). Bars show mean ± SEM. Scale bar, 5 μm.

Fig 3: TRIM67 alters exocytosis by modulating SNAP47 protein levels(A) Immunoblot of SNAP47 and GAPDH from lysate of Trim67+/+ and Trim67−/− cortical neurons at 2 DIV. Quantification of percent increase of SNAP47/GAPDH from 4 blots (right; log-paired t test, analysis in STAR methods).(B) Frequency of VAMP2-pHluorin exocytosis ± tagRFP or tagRFP-SNAP47 (n = 16 neurons per condition, n = 3 biological experiments, Welch’s t test).(C) Relative proportions of exocytic modes in Trim67+/+ neurons, Trim67−/− neurons, or Trim67+/+ neurons expressing tagRFP-SNAP47 (n = 16 neurons per condition; n = 3 biological replicates; multivariate linear regression, error bars report standard error of the percent).(D) tagRFP-SNAP47-normalized fluorescence intensity at fusion sites (n = 16 neurons, n = 3 biological replicates; Welch’s t test with Benjamini-Hochberg correction).(E) Quantification of time after peak ΔF/F before fluorescence decay onset of KNRd and FVFd events (n = 17 neurons; n = 4 biological replicates; Welch’s t test).(F) t1/2 of each exocytic class ± TagRFP-SNAP47 (n = 17 neurons per condition; Welch’s t test with Benjamini-Hochberg correction).(G) Measured neuronal surface areas at indicated times and conditions.(H) Example SNAP47 immunocytochemistry images in neurons expressing siRNA for SNAP47 knockdown (KD) (GFP+, arrow) or without (*). Cells were transfected with an eGFP expression plasmid and SNAP47 siRNA, followed by immunostaining of SNAP47; GFP-positive neurons did not stain for SNAP47, suggesting KD of SNAP47. (H’) Immunoblot and quantification of SNAP47 and βIII tubulin protein levels in SNAP47 KD compared with control (n = 3 experiments; Welch’s t test).(I) Frequency of VAMP2-pHluorin exocytosis ± SNAP47 siRNA (n = 12 neurons per condition; Welch’s t test).(J) Relative proportions of exocytic classes in Trim67+/+ neurons and Trim67−/− neurons, with SNAP47 KD (n = 12 neurons per condition, n = 3 biological replicates; multivariate linear regression, error bars report standard error of the percent).

Fig 4: Impact of Rarβ-silencing on β-cell function genes at protein level. Western blot analysis of Pro/insulin (A), PDX1 (B), NEUROD1 (C), p-AKT/AKT (D), GLUT2 (E), GCK (F), VAMP2 (G), and INSβ (H) normalized to the endogenous control protein β-actin. Corresponding fold changes in the intensity of the Western blot are shown below each blot. Data are obtained from three independent experiments. p-value. *: p < 0.05. Error bars show mean ± SD. Full western blot figures are available in Supplementary File.

Fig 5: VAMP7 forms a SNARE complex with SNAP-23 and Syntaxin11. (A) Model illustrating the formation of SNARE complex between VAMP7 (v-SNARE), SNAP-23, and Syntaxin11 (t- SNARE) during cytotoxic granule fusion in human CD8+ T cells. (B) Different Twin-Strep-tag fusion constructs used for pulldown assay. Twin-Strep-tag was fused at the C terminus of VAMP7 and at the N terminus of Syntaxin11 with a (GGS)x3 linker. (C) Western blot of bead stimulated human CD8+ T cells transfected with Twin-Strep-tag-tagged VAMP7, immuno-precipitated with anti-FLAG antibody and detected with antibodies against FLAG, SNAP-23, and Syntaxin11. (D) Western blot of bead stimulated human CD8+ T cells transfected with Twin-Strep-tag-tagged Syntaxin11, immuno-precipitated with anti-Syntaxin antibody and detected with antibodies against Strep-tag, SNAP-23, VAMP7, and VAMP2. As control, cells were transfected with Twin-Strep-tag construct. Ten percentage of the lysates were loaded as input. (E,F) Densitometric quantification of the Western blots shown in (C,D), respectively.