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. 1999 Jul 12;146(1):85–98. doi: 10.1083/jcb.146.1.85

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© 1999 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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The function of Vps33p is required early during the cell-free reconstitution system and precedes the function of Vam3p. (A) Experimental strategy. (B) Transport kinetics. A standard 50-μl reaction (see Fig. 3) was scaled up, incubated at 25°C, and then aliquots were removed and stopped at the indicated times. (C) Antibody inhibition. Aliquots from the same reactions as in A were also removed and received either 128 μg of immune IgG against Vps33p or 5 μl of antiserum against Vam3p (as indicated), incubated 15 min on ice, and then shifted to 25°C for 60 min. All reactions were immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography. Nonimmune serum was added to control for the effect of anti-Vam3p serum at each time point and did not inhibit the assay. A 2nd degree polynomial was used to fit the curve for the inhibition data points (C).