Fig. 3: SP9 inhibits triggered fusion in a reconstituted system.

a, The domain structure of Munc13-2 and its fragment (Munc13-2*). b, Single-vesicle content mixing assay with complete reconstitution (Methods). Stapled peptide (10 μM) was added together with SG vesicles and was present during all of the subsequent stages. c, The effect of SP9 on vesicle association. d, Corresponding Ca²⁺-independent fusion probabilities. e, Corresponding average probabilities of Ca²⁺-independent fusion events per second. For comparison, the result for the SR assay (Extended Data Fig. 7) is also shown. ***P = 0.00016. f, Corresponding Ca²⁺-triggered fusion probabilities at 500 µM and 50 µM Ca²⁺. g–i, Corresponding Ca²⁺-triggered fusion amplitudes of the first 1 s time bin after injection with 500 μM Ca²⁺ (g) (from left to right, ***P = 0.0000053, **P = 0.0024, **P = 0.0012); the cumulative Ca²⁺-triggered fusion probability within 1 min (h) (from left to right, ***P = 0.000058, **P = 0.0037, **P = 0.0026); and the decay rate (1/τ) of the Ca²⁺-triggered fusion histogram (i). The fusion probabilities and amplitudes were normalized to the number of analysed SG–airway PM vesicle pairs (Supplementary Table 2). For comparison, the results for the SR assay (Extended Data Fig. 7) are also shown. For c, e, g and h, box plots and data points are shown for n (indicated below each box plot) independent repeat experiments (Supplementary Table 2). For c, e, g and h, statistical analysis was performed using two-tailed Student’s t-tests. Decay constants (boxes) and error estimates (bars) in i were computed from the covariance matrix after fitting the corresponding histograms combining all repeats with a single exponential decay function using the Levenberg–Marquardt algorithm.

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