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. 2016 Sep 1;27(17):2697–2707. doi: 10.1091/mbc.E16-04-0229

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© 2016 Adolf 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: — The R-SNARE Sec22b and the Q-SNAREs Bet1 are present on the same vesicle membrane. (A) Schematic representation of homotypic COPII vesicle fusion. COPII vesicles reconstituted in the presence of GTP rapidly lose their coat after vesicle membrane scission, whereas vesicles reconstituted with the poorly hydrolyzable GTP analogue GTPγS remain coated and hence cannot undergo homotypic COPII vesicle fusion. (B) Formation of COPII vesicles was reconstituted in vitro by incubation of semi-intact HeLa cells with Sar1b, Sec23A/24A, or Sec23A/24C, Sec13/31A, and GTP or GTPγS plus an ATP-regenerating system (ATPr) as indicated and separated from donor membranes by medium-speed centrifugation. Vesicle fractions were analyzed by Western blotting for the presence of the non–vesicle membrane marker calnexin and the ER-Golgi SNARE proteins Sec22b and Bet1 as indicated. (Asterisks in B indicate immunostaining of immunoglobulin light chains from immunoprecipitated antibodies.)