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Protocols for Screening for Binding Partners of CCN Proteins: Yeast Two-Hybrid System

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CCN Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1489))

Abstract

Yeast two-hybrid screening is a powerful method to identify proteins that interact with a protein of interest. CCN2 consists of four domains, and identification of new proteins that bind to individual domains of CCN2 may reveal a variety of CCN2 functions. To identify CCN2-interactive proteins that regulate CCN2 activity, we carried out GAL4-based yeast two-hybrid screening with a cDNA library derived from a chondrocytic cell line, HCS-2/8, with CCN2 cDNA used as a bait. In this chapter, we describe our methods for screening for CCN2 binding partners by this system in detail. This protocol may be applied to other CCN proteins as well.

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References

  1. Young KH (1998) Yeast two-hybrid: so many interactions. (in) so little time …. Biol Reprod 58:302–311

    Google Scholar 

  2. Perbal B, Takigawa M (2005) CCN proteins: a new family of cell growth and differentiation regulators. Imperial College Press, London

    Book  Google Scholar 

  3. Takigawa M, Tajima K, Pan HO, Enomoto M, Kinoshita A, Suzuki F, Takano Y, Mori Y (1989) Establishment of a clonal human chondrosarcoma cell line with cartilage phenotypes. Cancer Res 49:3996–4002

    CAS  PubMed  Google Scholar 

  4. Keegan L, Gill G, Ptashne M (1986) Separation of DNA binding from the transcription-activating function of a eukaryotic regulatory protein. Science 231:699–704

    Article  CAS  PubMed  Google Scholar 

  5. Hoshijima M, Hattori T, Inoue M, Araki D, Hanagata H, Miyauchi A, Takigawa M (2006) CT domain of CCN2/CTGF directly interacts with fibronectin and enhances cell adhesion of chondrocytes through integrin alpha5beta1. FEBS Lett 580:1376–1382

    Article  CAS  PubMed  Google Scholar 

  6. Aoyama E, Hattori T, Hoshijima M, Araki D, Nishida T, Kubota S, Takigawa M (2009) N-terminal domains of CCN family 2/connective tissue growth factor bind to aggrecan. Biochem J 420:413–420

    Article  CAS  PubMed  Google Scholar 

  7. Hoshijima M, Hattori T, Aoyama E, Nishida T, Yamashiro T, Takigawa M (2012) Roles of heterotypic CCN2/CTGF-CCN3/NOV and homotypic CCN2-CCN2 interactions in expression of the differentiated phenotype of chondrocytes. FEBS J 279:3584–3597

    Article  CAS  PubMed  Google Scholar 

  8. Guthrie C, Fink GR (1991) Guide to yeast genetics and molecular and cell biology. Academic Press, Amsterdam

    Google Scholar 

  9. Dower WJ, Miller JF, Ragsdale CW (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 16:6127–6145

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

This work was supported by the programs JSPS KAKENHI Grants-in-aid for Scientific Research (B) No. JP15H05014 (to M.T.) and by Grant Nos. 22. 4293 and 26983161 from the Japan Society for the Promotion of Science (to M.H.).

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Authors and Affiliations

  1. Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama, 700-8525, Japan

    Mitsuhiro Hoshijima & Masaharu Takigawa D.D.S., Ph.D.

  2. Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama, 700-8525, Japan

    Takako Hattori

Authors
  1. Mitsuhiro Hoshijima
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  2. Takako Hattori
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  3. Masaharu Takigawa D.D.S., Ph.D.
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Corresponding author

Correspondence to Masaharu Takigawa D.D.S., Ph.D. .

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Editors and Affiliations

  1. Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama, Japan

    Masaharu Takigawa

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Hoshijima, M., Hattori, T., Takigawa, M. (2017). Protocols for Screening for Binding Partners of CCN Proteins: Yeast Two-Hybrid System. In: Takigawa, M. (eds) CCN Proteins. Methods in Molecular Biology, vol 1489. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6430-7_15

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  • DOI: https://doi.org/10.1007/978-1-4939-6430-7_15

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6428-4

  • Online ISBN: 978-1-4939-6430-7

  • eBook Packages: Springer Protocols

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