(E) Trans-SNARE Formation Assay: t- and v-SNARE SUVs were incubated in the presence or absence of different Munc18-1 constructs for 30min at 4°C. After solubilization and precipitation of the t-SNAREs using nickel beads, presence of full-length VAMP2 in the precipitates was probed by immunoblotting, which was used as an indicator for trans-SNARE assembly between SUVs. 