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We made recombinant baculovirus encoding glutathione-S-transferase (GST) fusion proteins to hChk1 (GST-hChk1) or to a mutation of hChk1 in which Asp at position 130 was mutated to Ala [GST-hChk1(D130A)] (pYS71). These recombinants were made by introducing an Nde I site at the first ATG codon of the hChk1 open reading frame using PCR, and subcloning the hChk1 cDNA as an Nde I-Eco RI fragment into pGEX2Tcs, generating pYS45. The Xba I-Eco RI fragment from pYS45 containing GST-hChk1 was subcloned into PVL1393, which was cut with Xba I and Eco RI, generating pYS63. The GST-hChk1 (D130A) mutant was generated by PCR, and the Xho I-Xmn I fragment containing the mutation was used to replace the wild-type fragment, generating pYS64. The GST-hChk1(D130A) fragment from pYS64 was sublconed into the baculovirus transfer vector by using the Univector plasmid fusion strategy (X. Liu and S. Elledge, unpublished results). Viruses were generated by standard methods (Baculogold, Pharmingen). Recombinant GST-hChk1 protein was isolated from infected Hi5 insect cells on glutathione (GSH) agarose. Cdc25C was cloned into pET15b (Novagen) and purified as outlined by the manufacturer.
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2-terminal sequencing. Manual Edman degradation was done as described (21) with a coupling and cleavage temperature of 55°C.
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We thank A. Baldini for assistance with mapping; T. Carr for sharing unpublished information; P. Sen for making the S216A mutants of Cdc25C; N. Walworth, W. Harper, M. Huang, and J. Bachant for helpful comments; and D. Leibham and J. Thompson for technical assistance. Support was by a NIH postdoctoral fellowship GM17763 to Y.S. and a NIH grant GM44664 to S.J.E. H.P.-W. is an Associate Investigator of the Howard Hughes Medical Institute. S.J.E. is a Pew Scholar in the Biomedical Sciences and an Investigator of the Howard Hughes Medical Institute.
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