Identification of connexin-interacting proteins: application of the yeast two-hybrid screen.
Protein-protein interactions are recognized as one of the fundamental mechanisms for relaying the intra- and intercellular signals that are required for normal cellular activities affecting growth, development, and maintenance of homeostasis in tissues and organs. The yeast two-hybrid screen has become a valuable tool for identifying protein-protein interactions. The gap junction protein connexin 43 (Cx43) has been implicated in a number of biological processes including development and cellular growth control. To further advance our understanding of the ways in which Cx43 may influence these cellular activities, and to extend our knowledge of the regulation of Cx43 function and/or processing, we have employed the yeast two-hybrid screen technique to identify Cx43-interacting proteins. We present detailed methods for the yeast two-hybrid screen of a mouse embryonic cDNA library using the C terminus of Cx43 as "bait." We also describe additional methods to confirm the interactions between Cx43 and the identified proteins. These methods include in vitro binding assays, coimmunoprecipitation, and subcellular localization using immunofluorescence microscopy.
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Molecular Carcinogenesis, Cancer Research Center of Hawaii, University of Hawaii at Manoa, 1236 Lauhala Street, Room 304, Honolulu, Hawaii 96813, USA.
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Martyn
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D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004622:Embryo, Mammalian; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D017629:Gap Junctions / Q000502:physiology; D015723:Gene Library; D018014:Gene Transfer Techniques; D051379:Mice; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D018031:Connexin 43; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors; D001616:beta-Galactosidase; D000099160:LexA Repressor Protein; C052130:GAL4 protein, S cerevisiae