Figure EV4. The association of synaptobrevin with the syntaxin1:SNAP25:Munc18‐1 complex does not displace Munc18‐1.

1. Fluorescence spectra showing FRET upon addition of the syntaxin1:SNAP25‐TR: Munc18‐1 complex to fluorescently labeled synaptobrevin (Syb‐OG). The decrease in the donor emission was accompanied by an increase in the acceptor emission (TR indicates Texas Red, and OG indicates Oregon Green).
2. The incubation of the syntaxin1:SNAP25:Munc18‐1 complex with synaptobrevin, followed by chemical cross‐linking resulted in an increase in the molecular size/hydrodynamic radius of the sample. The separation of the cross‐linked samples was performed on an analytical Superose 6 column (GE Healthcare). The cross‐linked sample after synaptobrevin incubation (red curve) showed a lower retention volume as compared to the one without synaptobrevin incubation (black curve), indicating continued association of Munc18‐1 after synaptobrevin binding.