All 3 recombinant VAMPs formed SDS-resistant ternary SNARE complexes in vitro.

Equimolar amounts of SNAP-25, syntaxin 4, and rVAMP-2, rVAMP-3, or rVAMP-8 were incubated together, and the samples were analyzed by SDS-PAGE and immunoblotting using antisyntaxin 4 primary antibodies. The incubation time, buffer composition, and temperature mimicked the conditions in the experiments studying the effects of rVAMPs on secretion in permeabilized platelets. Note that rVAMP-2, the negative control in the granule-secretion experiments, interacted readily with other SNAREs to form stable SNARE complexes in vitro.