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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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Wild-type MAPKK1 (0.5 μg) was incubated with LF (0.2 μg) as described in Fig. 2A for 30 min at 30°C. Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylidene difluoride membrane in CAPS transfer buffer {3-[cyclohexylamino]-1 propanesulfonic acid (10 mM, pH 11) and 10% methanol) at 300 mA constant current for 30 min. After transfer, membranes were quickly stained with Ponceau S solution (0.1% Ponceau S and 5% acetic acid) and rinsed with distilled deionized water. The appropriate band was cut from the membrane and subjected to automated Edman degradation in an Applied Biosystems 477A gas-phase sequencer, and phenylthiohydantoin derivatives were identified online with a 120 phenylthiohydantoin analyzer.
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2, 0.35 μg of MAPK [ERK2; D. J. Robbins et al., J. Biol. Chem. 268, 5097 (1993)], and 10 μg of MBP (Upstate Biotechnology)} was added, and samples were incubated for a further 15 min at 30°C. Proteins were separated by SDS-PAGE on 14% gels and processed for autoradiography.
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0027932676
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2+-charged HiTrap Chelating column (Pharmacia), washed with EB containing 10 mM imidazole, and eluted with EB containing 200 mM imidazole. Fractions containing recombinant protein were then pooled and desalted and concentrated in buffer containing 50 mM tris (pH 8.0), 0.1 M NaCl, 10% glycerol, 0.01% Triton X-100, and 1 mM DTT by centrifugation with a Centriprep-30 centrifugal concentrator (Amicon, Beverely, MA, XX).
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2642618551
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note
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This paper is dedicated to the memory of Ken Paull, whose untimely death is a significant toss to NCl and the cancer research community. The authors thank A. Cline and R. Frederickson for preparing the manuscript, L. Miller for technical assistance, H.-M. Koo for helpful discussions, M. Jeffers and S. Koochekpour for critical comments on the manuscript, and A. Murray for providing A90 cyclin. This research was sponsored in part by NCl, Department of Health and Human Services, under contract with Advanced BioScience Laboratories. By acceptance of this article, the publisher or recipient acknowledges the right of the U.S. government and its contractors and agents to retain a nonexclusive royalty-free license in and to any copyright covering the article.
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