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note
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2, 475 mM KCl, and 0.5% NP40. Ubiquitination assays with APC bound to antibody beads and elution of APC from the beads for SDS-polyacrylamide gel electrophoresis (PAGE) analysis were done as described (5). APC preparations from two different mitotic extracts (Δ90 extracts and extracts from metaphase II-arrested eggs) (5) had identical subunit compositions and ubiquitination activities.
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10544246978
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note
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2, and 100 μg/ml bovine serum albumin (BSA)] containing either 33 μg γ-PPase (300 units per microgram; Biolabs) or no γ-PPase. BSA and phosphatase were removed by washing the beads as described (9).
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10544248679
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note
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APC (∼250 μg) was purified from 500 ml of interphase Xenopus extract (15 g of protein) (9). Individual polypeptides were separated by SDS-PAGE. transferred to polyvinyldifluoride membrane (Bio-Rad) and processed for microsequencing at the Harvard Microchemistry Facility as described (6). APC1 and APC7 were digested with Lys-C, and APC3 and APC6 were digested with trypsin. The following sequences were obtained: APC1, DYIAPLPFQVANVXP, AEEQNAVLNLDQLGTPQHGMTTSSLTANLR, LSWTRNCDFEGSL, and PMTSIG; APC3, LLHLPAALGPLNPQFGIL, ILFANEK, and ALQELEELK; APC6, EFDFER, CYDFDVHTMK, and GLXLTAQY; APC7, EFFLAHIYTELQLIEEAL Only high confidence sequences are listed, except for the third APC1 sequence which is derived from an equimolar mixture of two peptides. Single-letter abbreviations for the amino acid residues are as follows A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln: R, Arg: S. Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
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10544226968
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note
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Membranes were isolated as described (5) with one additional washing step, and S100 and Resource Q column fractions were prepared as in (9). Fractions 13 to 15 (Fig. 2B) were pooled and further separated on a Superose 6 HR 10/30 column (Pharmacia) equilibrated in buffer Q-A (5). APC eluted with a molecular mass of 1.6 million daltons.
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note
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We thank P. Hieter, S. Tugendreich, A. Page, and T. Rapoport for antibodies: K. Nasmyth, W. Zachariae, and T. Shin for communicating unpublished results: H. Yu for recombinant UBCx; A. Georgi and T. Bernai for assistance with immunoblots; E. Brundell for help with the Tsg24 antibodies; and T. Enoch. M. O'Connell, and A. Amon for discussions. Supported by fellowships from the European Molecular Biology Organization (J.-M.P.) and by grants from the Swedish Cancer Society (C.H.), the Kjell and Marta Beijers Stiftelse (C.H.), and the National Institute of General Medical Sciences (M.W.K.).
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