(C) GUVs (14 nmol lipid) containing Syx1 (14 pmol) were pre-incubated with the indicated Munc18-1 constructs (90 pmol) at room temperature for 30 min. Subsequently, samples were incubated with soluble SNAP-25 (128 pmol) and SUVs (2.5 nmol lipid) containing Synaptotagmin1 (3 pmol) and SSynaptobrevin/VAMP2 (8 pmol) for 15 min on ice. Lipid mixing was started by increasing the temperature to 37 °C. 