Fig. 3
Low expression levels of Wt a-syn inhibit stimulated exocytosis of REs, whereas high expression levels cause enhancement, and mutants of a-syn show distinct differences. As indicated, RBL cells were co-transfected with VAMP8-pHluorin and low (5 μg a, b) or high (25 μg c, d) levels of pcDNA, Wt a-syn or a-syn mutants. VAMP8-pHluorin fluorescence increase was monitored in confocal movies (similar to Supplementary Movies 2a, b) before and after addition of thapsigargin (250 nM) to stimulate exocytosis, and after addition of NH4Cl (50 mM, 300–400 s after stimulation) to dequench intracellular VAMP8-pHluorin fluorescence. a Averaged exocytosis stimulated by thapsigargin in cells with low a-syn expression levels (n = 55); spontaneous release for each of these samples is less than 2.5% as shown in Supplementary Fig. 5. b Representative traces of VAMP8-pHluorin fluorescence integrated from movies for a of multiple confocal fields of 5–6 cells (all traces shown in Supplementary Fig. 5). c Averaged exocytosis stimulated by thapsigargin in cells with high a-syn expression levels (n = 55). d Representative traces of VAMP8-pHluorin fluorescence integrated from movies for c as in b (All traces shown in Supplementary Fig. 9). All data sets shown are from 3 to 4 independent experiments; Error bars are ±SEM; ***P-values < 0.001, **P-values < 0.01, *P-values < 0.05
