Tomosyn binds to the syntaxin monomer but does not inhibit assembly of the t-SNARE complex.
A, Coomassie Blue-stained SDS-PAGE gels showing the input materials of liposomes and proteins. Syn4, Syntaxin-4; Syn1, Syntaxin-1. B, Coomassie Blue-stained SDS-PAGE gel showing the binding of tomosyn and SNAP-23 to protein-free or syntaxin-4 liposomes. Syntaxin-4 liposomes were incubated with or without the recombinant tomosyn protein at 4 °C for 1 h, before SNAP-23 was added. After another hour of incubation at 4 °C, the samples were floated up on a Nycodenz gradient. C, Coomassie Blue-stained SDS-PAGE gel showing the binding of tomosyn and SNAP-25 to protein-free or syntaxin-1 liposomes. Each binding reaction contained 5 μm Syntaxin. Soluble factors were added to a final concentration of 5 μm.