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. 2000 Oct 30;151(3):551–562. doi: 10.1083/jcb.151.3.551

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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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Vps39 and Vps41 are components of the C-Vps complex. Spheroplasts expressing single-copy protein (prot) A or protein A–VPS39 were labeled with [³⁵S]methionine for 20 min and chased for 1 h at 30°C. Osmotic lysates of these strains were centrifuged at 100,000 g to generate the P _total. 0.5% NP-40/250 mM NaCl extracts of the P _total were incubated with IgG-sepharose to affinity-purify protein A fusions. Washed IgG-sepharose was analyzed by SDS-PAGE/fluorography. Multiple proteins coprecipitated preferentially with protein A–Vps39 (several are indicated by asterisks), but not protein A alone (lanes 1 and 2). IgG-sepharose–purified protein A fusions were also subjected to denaturation and immunoprecipitation using antibodies specific for Vps11, Vps18, Vps16, Vps33, and Vps41 (lanes 3–12).