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Rsk (Rsk). Primers 1 and 4 and 2 and 4 were used to create constructs ΔCT Rsk and CA Rsk, respectively. Polymerase chain reaction products were subcloned into pOTV-FLAC-LIC with a ligation-independent cloning method (Novagen, Madison, WI). With the T7 promoter, this vector directs the synthesis of mRNA coding for a FLAG epitope fusion protein of the inserted gene. Messenger RNA was synthesized for each of the constructs with the T7 message machine kit (Ambion, Austin, TX).
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0345470845
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note
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32P]ATP, and 250 μM S6 peptide (Upstate Biotechnology, Waltham, MA). Where indicated in Fig. 2, assays also contained 10 units of MAPK (New England Biolabs, Beverly, MA) or 2.5 ng of activated PDK-1 (Upstate Biotechnology). Reactions were incubated at 30°C for 60 min and quenched by the addition of EDTA to 60 mM. The beads were removed by centrifugation at 16,000g, and 10 μl of the supernatant were spotted onto p81 paper in duplicate, washed, dried, and counted by the Cerenkov method. Protein immunoblots were performed essentially as described elsewhere (3, 4).
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0344176941
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note
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Embryos were fertilized in vitro as previously described (4). Blastomeres of two-cell embryos were injected with 30 nl of mRNA (12.5 to 25 ng) encoding β-GaL, Mos, Rsk, ΔCT Rsk, or CA Rsk and then monitored with a dissecting microscope.
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0345470844
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note
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xe antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and antibody to cydin B2 were used as previously described (3).
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0344176920
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note
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Rsk (9) and E. Erikson for a critical reading of the manuscript. Supported by grant DK28353 from NIH. S.D.G. and M.S.S. are Associates and J.LM. is an Investigator of the Howard Hughes Medical Institute.
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