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. 2001 Feb 5;152(3):519–530. doi: 10.1083/jcb.152.3.519

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© 2001 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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Kinase-defective Vps15p is unable to form Vps30p complexes. AKY109 (Δvps34)/pKHR54 (VPS34) (lane 1), AKY109/pKHR60 (vps34-N736K) (lane 2), AKY115 (Δvps15)/pKHR55 (VPS15) (lane 3), and AKY115/pKHR59 (vps15-E200R) (lane 4) cells were grown to mid-log phase in SC medium lacking uracil at 28°C. Total lysates were solubilized with Triton X-100 and incubated with protein A–immobilized anti-Vps30p antibodies. Bound proteins were washed and eluted with 100 mM glycine-HCl, pH 2.5. Proteins were separated by SDS-PAGE, followed by detection by immunoblotting with anti-Vps30p, anti-Apg14p, anti-Vps34p, anti-Vps15p, and anti-Vps38p antibodies.