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note
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6 fusion. A 269-amino acid recombinant protein was expressed in Escherichia coli according to the protocol provided by the manufacture (Novagen). Recombinant protein was purified up to 90% with Ni-nitrilotriacetic acid (NTA) beads (Qiagen). Full-length hSecurin (or PTTG) was isolated with primers 5′-ATGGCTACTCTGATCTATGTTGATAAGG-3′ and 5′-TTAAATATCTATGTCACAGCAAACAGGTG-3′. The PCR product was then cloned into a modified version of pCS2 for in vitro translation and transfection into mammalian cells. The same fragment was also cloned into a modified pET28a vector to express recombinant protein in E. coli. Protein more than 90% pure was obtained by affinity purification with Ni-NTA beads (Qiagen) followed by Resource Q column chromatography. Rabbit polyclonal antibodies to the 269-amino acid hESP1 fragment and full-length hSecurin (or PTTG) were made by Zymed with purified recombinant proteins as antigen. Crude sera were affinity-purified by passing them through affinity AffiGel-10 (BioRad) columns coupled with corresponding antigens. Antibodies were eluted from the affinity-columns by 100 mM glycine (pH 2.5) followed by 100 mM triethylamine (pH 11.5). Affinity-purified antibody to hESP1 (anti-hESP1) cross-reacts with Myc tag. This antibody does not bind to in vitro-translated hSecurin (24). The anti-cyclin B1 used in this study was a rabbit antibody purchased from Santa Cruz Biotechnology.
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0345197499
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2-M (2N), and the percentage for each stage of the cell cyde was calculated.
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17
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0344334997
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note
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2, 1 mM EDTA. 1 mM dithiothreitol, insulin (0.1 mg/ml), and 10% glycerol (same below); once in 300 mM KCI and 0.5% NP-40 wash buffer; and once in 500 mM KCl and 0.5% NP-40 wash buffer.
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18
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1842367945
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25
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0344767043
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note
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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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33
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0344767042
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note
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Extract prepared from nocodazole-arrested HeLa S3 celts was fractionated on a Resource Q column with a KCl concentration gradient from 100 mM to 1 M in the lysis buffer. Human ESP1 was eluted at about 250 mM KCl. Fractions containing hESP1 were pooled and loaded onto a Superdex 200 column.
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34
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0032133145
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Yu, H.2
M, W.K.3
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35
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0344767041
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note
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We thank C. Pfleger and S. Rankin for comments on the manuscript G. Fang, H. Yu, and other members of the Kirschner laboratory for helpful discussion and technical assistance; C. Pfleger and E. Lee for providing modified pCS2 and pET28a vectors; and Q. Zang for mammalian cell transfection. H.Z. is a fellow of The Jane Coffin Childs Memorial Fund for Medical Research. T.J.M. was supported by a Physician Post-doctoral Grant from the Howard Hughes Medical Institute and a Mentored Clinical Scientist Development Award from the National Heart, Lung, and Blood Institute. Supported by grants GM39023 and GM26875 from NIH awarded to M.W.K.
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