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. 2008 Nov 3;183(3):499–511. doi: 10.1083/jcb.200806016

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© 2008 Semerdjieva 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 1. — Rab5 specifically regulates AP2 uncoating in vitro. (A) Uncoating assays were performed on CCVs isolated from porcine brain incubated with ATP, Hsc70 (1.3 μg), and cytosols (50 μg) prepared from HEK293T cells overexpressing Rab5^wt, Rab5^Q79L, Rab5^S34N, or Rab1^wt as indicated. (top) CCVs (P) were separated from released coat proteins (S) and analyzed by Western blotting for clathrin using CHC 5.9, AP2 using anti–α-adaptin mAb 100/2, and AP1 using anti–γ-adaptin mAb 100/3. (bottom) α-Adaptin, clathrin, and γ-adaptin amounts released into supernatant during uncoating quantified by densitometry of Western blots (n = 3). Data expressed as a percentage ± SEM of maximal uncoating seen in the presence of rab5^wt or rab5^Q79L. AP2 uncoating in the presence of rab5^S34N and rab1^wt was significantly different from uncoating by rab5^wt (**, P < 0.01) and rab5^Q79L (***, P < 0.001). (B, top) HEK293T cytosol preincubated with anti-rabaptin5 antibodies to deplete rabex-5 or with nonspecific IgG was assayed for its ability to promote clathrin, AP2, and AP1 uncoating. (bottom) α-Adaptin present in supernatant was quantified and is expressed as a percentage of coat protein uncoating after treatment of cytosol with nonspecific IgG. Results are the mean ± range of duplicate samples. (C, top) Uncoating assays were performed in the presence of HEK293T cytosol or HEK293T cytosol overexpressing rab5^wt or hRME-6. (bottom) α-Adaptin present in supernatants was quantified and expressed as a percentage of AP2 uncoating after incubation with cytosol overexpressing HA–hRME-6. Results are means ± range of duplicate samples. (D, top) Cytosol overexpressing hRME-6 was treated with anti-rabaptin5 antibodies or nonspecific IgG and assayed by Western blotting for its ability to uncoat AP2. (bottom) AP2 present in supernatants was quantified and expressed as a percentage of AP2 uncoating after incubation with control cytosol. Results are means ± range of duplicate samples. (E) Uncoating assays were performed in the presence of increasing concentrations of PP2A and supernatants were assayed for release of AP1 and AP2.