Prediction-driven in vitro experiments and analysis of neuronal SM-SNARE network motif. (a) and (b) lipid mixing assay of wildtype neuronal SM-SNARE system with different initial concentration configurations of Munc18-1. The result confirms the bifurcation behavior predicted by simulation experiments (shown in Figure 3) that when the concentration of Munc18-1 is equal to the concentration of Syntaxin-1 the fusion effect reaches maximum (purple line). (c) and (d) Mutant lipid mixing assay of neuronal SM-SNARE system with separately expressed SNAP2 and mutant syntaxin-1. The result of b and c confirmed that the complicated bifurcation behavior is introduced by the unique binding mode between Munc18-1 and Syntaxin-1 because the mutant system which deletes the binding mode shows a similar behavior to the yeast system without bifurcation phenomenon.