Extended Data Fig. 7: Related to Figure 3. SP9 inhibits Ca²⁺-triggered vesicle fusion with reconstituted airway epithelial SNAREs and Syt2.

a, Schematic of the single vesicle content mixing assay. Airway PM: plasma membrane mimic vesicles with reconstituted airway Stx3 and SNAP-23; SG: secretory granule mimics with reconstituted VAMP8 and Syt2. After SG - airway PM vesicle association, vesicle pairs either undergo Ca²⁺-independent fusion or remain associated until fusion is triggered by Ca²⁺ addition. 10 μM of P0 or SP9 was added together with SG vesicles and was present during all subsequent stages. b, Effects of P0 or SP9 on vesicle association. c, Corresponding Ca²⁺-independent fusion probabilities. d, Corresponding average probabilities of Ca²⁺-independent fusion events per second (* p = 0.014). e, Corresponding Ca²⁺-triggered fusion probabilities. (f–h) Corresponding Ca²⁺-triggered fusion amplitudes of the first 1-sec time bin upon 500 μM Ca²⁺-injection (f) (* p = 0.012), the cumulative Ca²⁺-triggered fusion probability within 1 min (g) (** p = 0.0018), and the decay rate (1/τ) of the Ca²⁺-triggered fusion histogram (h). i–k, SP9 has no effect on vesicle fusion mediated by airway SNAREs alone. i, Effects of 10 μM of P0 or SP9 on vesicle association using the assay described above. j, Corresponding Ca²⁺-independent fusion probabilities. k, Corresponding average probabilities of Ca²⁺-independent fusion events per second. Panels b, d, f, g, i, k show box plots and data points for n (indicated below each box plot) independent repeat experiments (Supplementary Table 2). Two-tailed Student’s t-tests were used for SP9 vs. No SP. Decay constants (boxes) and error estimates (bars) in panel h computed from the covariance matrix upon fitting the corresponding histograms combining all repeats with a single exponential decay function using the Levenberg-Marquardt algorithm.

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