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2, and stored at -80°C. Purified p115 was >99% pure as determined by Coomassie blue staining. Before use, p115 protein was pelleted at 14,000 rpm for 10 min at 4°C to remove precipitated material.
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7 M500 Dynabeads (Dynal, New York) coupled to p115 mAb 3A10. Immunoprecipitation was carried out for 2 hours at 4°C with rotation. Beads were washed four times for 5 min each with RIPA buffer without SDS, and bound proteins were eluted by boiling in SDS sample buffer supplemented with 10 mM DTT for 5 min. Amounts of SNAREs and SNARE-binding proteins recovered in the p115 complex on COPII vesicles (Fig. 4A, lane b) were determined by densitometry of immunoblots. Recovery was expressed as a percentage of the amount of specific protein recovered in the HSP of the normal in vitro incubation minus the background levels observed in the HSP in the control (Sar1-GDP-containing) incubation that prevents COPII vesicle formation. In the microsome control, the level of p115-SNARE associations was less than 0.1%.
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We thank G. Waters for p115 cDNA and p115 mAbs; G. Warren for p97 and p47 antibodies; R. Scheller for rbet1, membrin, and sec22 cDNAs; H. Plutner for excellent technical assistance; and P. Tan for help during the initial phase of this work. Supported by NIH grants GM 33301 and GM42336 and National Cancer Institute grant CA58689 (W.E.B.), a NIH National Research Service Award (B.D.M.), and a Wellcome Trust International Traveling Fellowship (B.B.A.).
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