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Interaction of S100A6 with Target Proteins In Vitro and in Living Cells

  • Kyohei Sakane
  • Fuminori Yamaguchi
  • Mitsumasa Tsuchiya
  • Rina Kondo
  • Naoki Kanayama
  • Masaki Magari
  • Naoya Hatano
  • Ryoji Kobayashi
  • Hiroshi TokumitsuEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1929)

Abstract

S100A6 is a member of the EF-hand Ca2+-binding protein family, which plays important roles in a wide variety of Ca2+ signaling in the cells, as well as in pathophysiological conditions. Herein, we describe analytical protocols for evaluating the interaction of S100A6 with multiple target proteins in vitro, including biotinylated S100A6 overlay, glutathione-S-transferase (GST)-precipitation, surface plasmon resonance, and a GST-precipitation assay in living cells. These methods will elucidate the detailed molecular mechanisms of S100A6/target interactions and further improve our understanding of the physiological significance of S100A6-mediated Ca2+ signaling. Moreover, they may be used to evaluate other physical S100/target interactions.

Key words

S100A6 Ca2+-binding EF-hand Protein–protein interaction FOR20 Protein phosphatase 5 GST-precipitation assay S100A6-overlay Transfection SPR analysis 

Notes

Acknowledgment

This work was supported in part by a grant (to H.T.) from the Ryobi Teien Memory Foundation, Japan.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Kyohei Sakane
    • 1
  • Fuminori Yamaguchi
    • 2
  • Mitsumasa Tsuchiya
    • 3
  • Rina Kondo
    • 1
  • Naoki Kanayama
    • 1
  • Masaki Magari
    • 1
  • Naoya Hatano
    • 1
  • Ryoji Kobayashi
    • 4
  • Hiroshi Tokumitsu
    • 1
    Email author
  1. 1.Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health SystemsOkayama UniversityKita-kuJapan
  2. 2.Ehime Prefectural University of Health SciencesIyo-gunJapan
  3. 3.Emergency and Critical Care Center, Kawasaki Municipal Kawasaki HospitalKawasakiJapan
  4. 4.Sakuragi Psychiatric HospitalMimaJapan

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