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. 2008 Sep 22;182(6):1141–1151. doi: 10.1083/jcb.200801001

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© 2008 Brett et al.

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.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 3. — Native Gyp7 inactivates Ypt7p in vivo. (a) Vacuoles isolated from BJ3505 wild type (GYP7) or gyp7Δ were incubated with ATP for 40 min with 9.5 μM rGdi1 absent or present during the last 10 min. Vacuoles were sedimented, and membrane association of Vps41, Vam3, and Ypt7 was assessed by immunoblotting. The white line indicates that intervening lanes have been spliced out. (b) Vacuoles isolated from BJ3505 wild-type (GYP7, top) or gyp7Δ cells (bottom) were pretreated with or without 200 μM GTPγS for 10 min at 27°C and incubated for 40 min with ATP in the presence or absence of 3.6 μM rGyp1_TBC at four concentrations of sorbitol (standard conditions). During the last 10 min of incubation, 9.5 μM rGdi1 was added. Vacuoles were immediately sedimented, and the membrane distribution of Ypt7 was assessed by immunoblotting. One third of the total isolated supernatant (S) and one fifth of the pellet (P) were analyzed by immunoblotting.