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note
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2-terminal domain of human IQGAP1 (amino acids 1 to 216).
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3542998338
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note
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2, and the lysates were incubated for 2 hours at 4°C in the absence or presence of the indicated antibodies. The resulting immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis with the indicated antibodies. About 50% of IQGAP1 was immunoprecipitated from EL cells incubated in the absence or presence of DSP (13).
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3543039079
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unpublished data
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S. Kuroda et al., unpublished data.
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Kuroda, S.1
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3543018749
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note
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Complementary DNAs encoding mouse α-catenin and mouse β-catenin, as well as a cDNA fragment encoding the cytoplasmic domain of mouse E-cadherin, were inserted individually into the Bam HI site of pMAL C-Z. The encoded MBP fusion proteins were expressed in and purified from Escherichia coli. GST-IQCAP1 was purified from overexpressing insect cells as described (23). Various concentrations of MBP fusion proteins were mixed with affinity beads coated with GST or GST-IQCAP1. The beads were washed, and bound MBP fusion proteins were coeluted with GST or GST-IQCAP1 by the addition of reduced glutathione. The eluates were subjected to SDS-PAGE and immunoblot analysis with antibodies to MBP (New England Biolabs, Beverly, MA).
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3543015232
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note
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Various concentrations of MBP-α-catenin and MBP-β-catenin were mixed and incubated for 1 hour at 4°C. The mixture was then incubated with beads coated with GST, GST-IQGAP1, or GST-E-cadherin (the cytoplasmic domain). The interaction of MBP fusion proteins with GST fusion proteins was examined (14).
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3543024155
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note
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GST-1QGAP1-N and GST-IQGAP1-C were produced and purified as described (23). GST-IQGAP1-ΔC was purified from overexpressing insect cells. MBP-β-catenin (50 nM) or MBP-E-cadherin (250 nM) was mixed with affinity beads coated with the indicated GST fusion proteins (40 pmol each). The beads were washed, and the interaction of MBP fusion proteins with GST fusion proteins was examined (14).
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3543018750
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note
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The plasmids pEF-BOS-Myc-IQGAP1, pEF-BOS-Myc-IQGAP1-N, pEF-BOS-Myc-IQGAP1-C, and pEF-BOS-Myc-IQGAP1-ΔC were constructed (9, 13) and microinjected (0.01 mg/ml) into nuclei of subconfluent EL cells. After 5 hours, the cells were fixed and doubly stained with antibodies to Myc and to β-catenin. Similar results were obtained by transfection (13).
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Itoh, N.1
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note
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2+ or EGTA as described (3).
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3543033063
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note
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6) were transferred to six-well culture dishes (19), and, after incubation for 24 hours, they were treated with DSP (12) and lysed. The lysates were centrifuged at 100,000g for 30 min, and the resulting supernatant was mixed with antibodies to E-cadherin (Transduction Laboratories). After incubation at 4°C for 1 hour, the immunoprecipitate was separated by centrifugation and subjected to SDS-PAGE and immunoblot analysis with the indicated antibodies.
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note
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We thank A. Nagafuchi and S. Tsukita for providing L, EL, and nEαCL cells; cDNAs encoding E-cadherin, α-catenin, or β-catenin; and antibodies to E-cadherin (ECCD-2) or to α-catenin. We also thank M. Takeichi for providing ECCD-2 and Kazusa DNA Research Institute for support of a cDNA Research Program. Supported by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (1997), by the Japan Society for the Promotion of Science Research for the Future, by the Human Frontier Science Program, and by grants from Kirin Brewery Co. Ltd.
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