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. 2011 Mar 1;22(5):624–633. doi: 10.1091/mbc.E10-06-0484

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© 2011 Orlando et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 4: — Analysis of Cdc42p association with different classes of secretory vesicles by Percoll density gradients. The sec10-2 mutant cells, which have block in both Bgl2p and invertase secretion, were used to analyze Cdc42p association with vesicles. A sec10-2 mutant strain with a copy of HA-Cdc42p integrated at its URA3 locus on the chromosome was used for membrane preparation as described in Materials and Methods. The secretory vesicle preparation was then subjected to Percoll density gradients. The collected fractions were analyzed by Western blot to measure the distributions of HA-Cdc42p (A) and Bgl2p (B) or tested for invertase activity (C). Two major peaks in the HA-Cdc42p blot were observed: one around fractions 5–9, which corresponds to the lighter Bgl2p vesicles, and the other around fractions 14–17, which corresponds to denser invertase vesicles.