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DH-Sos DNA was generated by polymerase chain reaction (PCR) amplification of hSos1 template with oligonucleotides 5′-CCGGTTAACGTAGAAGATATTAATATA-3′ and 5′-CCGGGATCCCTAATTACAACACTGTCCAATGTC-3′, which correspond to the sequences between nucleotide positions 538 to 555 and 1306 to 1326, respectively, of hSos1. The amplified fragment was ligated into T7 epitope-tagged pCMG6KR mammalian expression vector. DH-PHSos1 was generated by PCR amplification of a hSos1 template using oligonucleotides 5′-CCGTCTAGAGTAGAAGATATTAAT-3′ and 5′-CAAGGTACCTCATTCCAGTGTACTCCG-3′, which correspond to the sequences between nucleotide positions 538 to 552 and 1639 to 1653, respectively, of hSos1. The amplified fragment was ligated into HA epitope-tagged pCGN mammalian expression vector. Other expression vectors used in this study are described in (6).
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15
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14444281034
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note
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32P]ATP (7000 Ci/mmol) (ICN). The reaction products were resolved on SDS-polyacrylamide gels, visualized by autoradiography, and quantified with a Phosphorlmager.
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17
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14444267701
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note
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2]. The GST fusion proteins were isolated by precipitation with glutathione Sepharose and bound nucleotides were eluted with 1% SDS and 20 mM EDTA for 5 min at 65°C. Radioactivity was quantified by scintillation counting.
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18
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personal communication
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4] were microinjected into cell nuclei. Three hours after injection, cells were fixed with 3.7% formaldehyde in phosphate-buffered saline for 1 hour, then permeabilized with 0.1 % Triton X-100 for 3 min, and incubated for 1 hour with rhodamine-conjugated phalloidin (Molecular Probes). The cells were photographed with a Zeiss Axiovert microscope.
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14444274473
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note
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Proteins were fractionated by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membrane (Schleicher & Schuell) at a constant voltage of 90 V for 45 min in transfer buffer [25 mM tris-HCl, 192 mM glycine, 20% (v/v) methanol]. The membranes were incubated with monoclonal antibody to T7 (Novagen), monoclonal antibody to HA (12CA5) (Babco), monoclonal antibody to Myc (9E10), or polyclonal antibody to JNK1 (Santa Cruz Biotechnology) for 1 hour and then with horseradish peroxidase-conjugated goat antibody to mouse immunoglobulin G (IgG) or goat antibody to rabbit IgG (Cappel). The immunoreactive bands were visualized with the enhanced chemiluminescence detection system (Dupont-NEN).
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36
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14444270284
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note
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We thank A. Hall and R. Cerione for plasmids. Supported by National Institutes of Health grants CA28146 and CA55360. A.S.N. and B.A.Y. are supported by National Institutes of Health training grant CA09176.
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