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. 2000 Jan 24;148(2):247–252. doi: 10.1083/jcb.148.2.247

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© 2000 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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Association between nSec1 and syntaxin prevents toxin-resistant SNARE complex formation. Either syntaxin1A (SYN1; lanes 1, 2, 5, and 6) or nSec1/syntaxin1A complex (SYN1/nSEC1; lanes 3, 4, 7, and 8) was combined with full-length SNAP-25 (SN25) and VAMP2. The mixtures were incubated for 1 h at 4°C. Then, either botulinum toxin E to digest SNAP-25 (BotT E; upper panel of lanes 5–8) or botulinum toxin D to digest VAMP2 (BotT D; lower panel of lanes 5–8) was added and incubated 30 min at 25°C. After incubation, 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. Proteins were then separated on a 16% SDS-polyacrylamide gel and visualized by Coomassie blue staining.