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Immunoblot Analysis of Linear Polyubiquitination of NEMO

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NF-kappa B

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1280))

Abstract

Stimulation with inflammatory cytokines such as TNF-α and IL-1 activates the canonical NF-κB pathway through the activation of the IKK complex. The mechanism underlying IKK activation has been extensively studied and the involvement of the ubiquitin system has been well documented. We have recently reported that a novel ubiquitin ligase complex, LUBAC is involved in the activation of the IKK complex. LUBAC consists of one catalytic subunit, HOIP and two accessory molecules, HOIL-1L and SHARPIN and activates the IKK complex by conjugating the linear polyubiquitin chains to NEMO (IKKγ), the regulatory subunit of IKK complex. In this chapter, we describe the protocol for the detection of the linear polyubiquitination of NEMO by the immunoblotting using anti-linear ubiquitin antibody.

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Acknowledgments

This work is partly supported by the Targeted Proteins Research Program (TPRP) and grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan to K.I. and/or Y.S.

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Authors and Affiliations

  1. Department of Molecular and Cellular Physiology, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501, Japan

    Yoshiteru Sasaki, Hiroaki Fujita, Misa Nakai & Kazuhiro Iwai

Authors
  1. Yoshiteru Sasaki
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  2. Hiroaki Fujita
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  3. Misa Nakai
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  4. Kazuhiro Iwai
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Corresponding author

Correspondence to Kazuhiro Iwai .

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Editors and Affiliations

  1. Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania, USA

    Michael J. May

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Sasaki, Y., Fujita, H., Nakai, M., Iwai, K. (2015). Immunoblot Analysis of Linear Polyubiquitination of NEMO. In: May, M. (eds) NF-kappa B. Methods in Molecular Biology, vol 1280. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2422-6_17

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  • DOI: https://doi.org/10.1007/978-1-4939-2422-6_17

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2421-9

  • Online ISBN: 978-1-4939-2422-6

  • eBook Packages: Springer Protocols

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