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3
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14444277560
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note
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Myc-epitope-tagged Cdc25C and Cdc25(S216A) were subcloned into pUHD10-3 (25). Plasmids were cotransfected with pBabe, a plasmid encoding a puromycin resistance gene, into the HeLa tTA cell line (23). Clones resistant to G418 (geneticin, Gibco) and puromycin were screened for inducible expression of Myc-tagged Cdc25C or Cdc25(S216A). Clones were expanded in Dulbecco's minimum essential medium (DMEM) containing G418 (400 μg/ml), puromycin (1 μg/ml), and tetracycline (2 μg/ml). Cells were trypsinized and washed four times with warm DMEM lacking tetracydine to induce protein expression. Upon replating, cells were grown in DMEM containing G418 and puromycin. Indirect immunofluorescence indicated that 85 to 90% of cells were induced to express Cdc25C and Cdc25(S216A) at levels 10-to 50-fold as high as endogenous Cdc25C (4). In some cases, cells were subjected to a doublethymidine block (7) or were incubated during the last 8 to 16 hours of induction with nocodazole (0.15 μg/ml) (Calbiochem) followed by mechanical agitation. Cells were lysed in mammalian cell lysis buffer [50 mM tris (pH 8.0), 2 mM dithiothreitol (DTT), 5 mM EDTA, 0.5% NP-40, 100 mM NaCl, 1 μM microcystin, 1 mM sodium orthovanadate, 2 mM phenylmethylsulfonyl fluoride (PMSF), aprotinin (0.15 U/ml), 20 μM leupeptin, and 20 μM pepstatin]. Antibodies used for Cdc25C detection included a monoclonal antibody to the Myc epitope (9E10 myc-agarose, Santa Cruz Biotechnology), a monoclonal antibody generated to Cdc25C (174E10-3), and an affinity-purified rabbit polyclonal antibody to glutathione-S-transferase (GST). 14-3-3 proteins were detected with antibody to 14-3-3 β (K-19, Santa Cruz), which is broadly reactive with members of the 14-3-3 family of proteins. Bound primary antibodies were detected with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Cappel) and an ECL detection system (Amersham).
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4
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14444284888
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C.-Y. Peng and H. Piwnica-Worms, unpublished results
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C.-Y. Peng and H. Piwnica-Worms, unpublished results.
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5
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14444274648
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note
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3 (pH 8.0). Proteolysis and two-dimensional phosphopeptide mapping were performed as described (24).
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6
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14444283688
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note
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9) were suspended in 5 ml of cell dissociation solution (Sigma Chemical) and elutriated at 25°C with a Beckman elutriator rotor (model J2MI) in RPMI medium containing 1% fetal bovine serum. Cells were processed for flow cytometric analysis as described (25) with a Becton-Dickinson FACScan, and data were analyzed with CELL QUEST software.
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9
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14444279047
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note
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Uninduced and induced cells were incubated for 8 hours in the presence of nocodazole (0.15 μg/ml) and then processed for chromosome spreading as described (8). For monitoring the DNA replication checkpoint, we incubated cells in the presence of 1 mM hydroxyurea (Sigma Chemical). After 8 hours, nocodazole was added to a final concentration of 0.15 μg/ml. After an additional 8 hours, cells were collected and analyzed for amounts of Cdc25C by immunoblotting, for cyclin B1-associated histone H1 kinase activity as described (7), and for chromosome integrity as described (8). For monitoring the DNA damage checkpoint, we labeled cells for 60 min with 20 μM bromodeoxyuridine (BrdU, Amersham) and then treated them with 0 or 6 Gy of gamma irradiation. Cells were harvested at 0, 4, 8, 12, 16, and 24 hours after irradiation, stained with propidium iodide (PI), stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies to BrdU, and processed for flow cytometric analysis as described (26). Because induction of the Cdc25(S216A) mutant protein occurred more quickly than for wild-type Cdc25C, inductions of Cdc25C were initiated at least 16 hours before Cdc25(S216A) inductions. In every experiment, lysates were prepared and monitored for amounts of Cdc25C and Cdc25(S216A) by immunoblotting, and in each case the Cdc25 (S216A) mutant accumulated to slightly smaller amounts than did wild-type Cdc25C.
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10
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0029871708
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A. J. Muslin, J. W. Tanner, P. M. Allen, A. S. Shaw, Cell 84, 889 (1996).
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Muslin, A.J.1
Tanner, J.W.2
Allen, P.M.3
Shaw, A.S.4
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12
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0030584088
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J. Zha, H. Harada, E. Yang, J. Jockel, S. J. Korsmeyer, Cell 87, 619 (1996).
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Zha, J.1
Harada, H.2
Yang, E.3
Jockel, J.4
Korsmeyer, S.J.5
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13
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14444278126
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note
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Stocks (1 mM) of phosphorylated (GLYRSPpSMPENLNRPR) (E, Glu; G, Gly; L, Leu; M, Met; N. Asn; Y, Tyr) and unphosphorylated peptides consisting of amino acids 210 to 225 of Cdc25C were prepared in 50 mM sodium phosphate, pH 7.0, and 0.5% sodium azide (vehicle). Lysates of Sf9 insect cells overproducing GST-Cdc25C were incubated with glutathione agarose beads. Beads containing Cdc25C: 14-3-3 complexes were washed three times with buffer A [1 × phosphate-buffered saline, 1% NP-40, 50 mM NaF, 5 mM EDTA, 2 mM DTT, 1 μM microcystin, 2 mM PMSF, 20 μM leupeptin, 20 μM pepstatin, and aprotinin (0.15 U/ml)], and peptides were added at the indicated concentrations in a final volume of 200 μl of buffer A. Incubations were carried out at 4°C for 1 hour followed by three washes with buffer A. Reactions were resolved by SDS-PAGE, and GST-Cdc25C and 14-3-3 were visualized by immunoblotting with GST and K-19 antibodies, respectively.
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14
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14444284643
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P. R. Graves and H. Piwnica-Worms, unpublished results
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P. R. Graves and H. Piwnica-Worms, unpublished results.
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16
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0028101138
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J. C. Ford et al., Science 265, 533 (1994).
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Science
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Ford, J.C.1
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20
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14444270425
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note
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2-terminal sequencing. Manual Edman degradation was performed as described (27) with a coupling and cleavage temperature of 55°C.
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21
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14444285110
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R. S. Thoma and H. Piwnica-Worms, unpublished results
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R. S. Thoma and H. Piwnica-Worms, unpublished results.
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22
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0030867582
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Y. Sanchez et al., Science 277, 1497 (1997).
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Sanchez, Y.1
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24
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M. Xu, K.-A. Sheppard, C.-Y. Peng, A. S. Yee, H. Piwnica-Worms, Mol. Cell. Biol. 14, 8420 (1994).
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Xu, M.1
Sheppard, K.-A.2
Peng, C.-Y.3
Yee, A.S.4
Piwnica-Worms, H.5
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27
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0025775736
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J. E. Bodwell et al., J. Biol. Chem. 266, 7549 (1991); S. Sullivan and T. W. Wong, Anal. Biochem. 197, 65 (1991).
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Lu, K.P.1
Hunter, T.2
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31
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14444275366
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note
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We thank M. J. Byrnes for technical support, the laboratory of S. Dowdy for assistance with fluorescence-activated cell sorting analysis, T. Carr and T. Enoch for S. pombe Chk1 reagents, and T. Enoch for insightful discussions. Supported in part by NIH grants GM47017 (to H.P.-W.) and AI34094 (to A.S.S.) and training grant GM18428 (to P.R.G.). H.P.-W. is an Associate Investigator of the Howard Hughes Medical Institute.
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