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. 2000 Mar 20;148(6):1223–1230. doi: 10.1083/jcb.148.6.1223

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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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Vam2/6p is required for trans-SNARE pairing. The formation of trans-SNARE pairs between vacuoles was assayed as described in Ungermann et al. 1998. Vacuoles from strains BJ3505nyv1Δ and DKY6281vam3Δ (100 μg each) were incubated together at 27°C (lanes 2–4) or on ice (lane 1) with cytosol and ATP for 30 min. Reactions were supplemented with control buffer (lanes 1 and 2), affinity-purified anti-Vam2p IgG (lane 3), or affinity-purified anti-Vam2p Fab fragment (lane 4). Protein complexes were analyzed by coimmunoprecipitation with protein A–immobilized antibodies to Vam3p, SDS-PAGE, and immunoblotting with antibodies to Vam3p and Nyv1p.