Figure 3.

nSec1 does not bind the ternary SNARE complex. Both GST and GST-nSec1 (nSEC1) were purified by affinity chromatography. Ternary SNARE complex (marked by an asterisk) was formed by mixing syntaxin1A, full-length SNAP-25, and VAMP2, and purified by size-exclusion chromatography. The purified GST or GST-nSec1 was incubated with glutathione-agarose beads for 1 h and washed. The beads were then incubated with purified ternary SNARE complex overnight at 4°C. The beads were washed and sample buffer (final concentration of 2% SDS) was added, and half of the mixture was boiled whereas the other half was kept at room temperature. Protein were separated on a 16% SDS-polyacrylamide gel. Molecular mass markers are indicated on the left in kD.