(B) GST-Munc18-1 constructs were immobilized on glutathione beads and incubated with the indicated molar excess of Synaptobrevin/VAMP2 (over Munc18-1) for 1.5 h at 4°C. Bound Synaptobrevin/VAMP2 was analyzed by Western blot and immune decorating with an anti-Synaptobrevin/VAMP2 antibody and quantified by the LICORE system and ImageJ software. Synaptobrevin/VAMP2 bound to the GST-Munc18-1 mutants is represented as percentage of Synaptobrevin/VAMP2 bound to GST-Munc18-1 WT. Error bars indicate s.e.m., n=3. 