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NF-kappa B pp 121–154Cite as

Systematic Detection of Noncanonical NF-κB Activation

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

Abstract

In unstimulated cells, NF-κB dimers usually exist as latent complexes in the cytoplasm with the IκB (inhibitor of NF-κB) proteins or IκB-like protein p100, the precursor of NF-κB2 mature form p52. Accordingly, there are two major mechanisms leading to NF-κB activation: inducible degradation of IκBs and processing of p100 to generate p52 (selective degradation of the C-terminal IκB-like sequence of p100), which are termed the canonical and noncanonical NF-κB pathways, respectively. While activation of the canonical NF-κB pathway plays critical roles in a wide range of biological processes, the noncanonical NF-κB pathway has important but more restricted roles in both normal and pathological processes. Systematic detection of the noncanonical NF-κB pathway activation is very important for understanding the physiological role of this pathway in biological processes, and for the diagnosis, prevention, and treatment of related diseases. We describe here the methods we employ to detect noncanonical NF-κB activation in cells and tissues. These methods are immunoblotting, co-immunoprecipitation, immunofluorescence, immunohistochemistry, chromatin immunoprecipitation (ChIP) analysis, and electrophoretic mobility shift assay (EMSA). Noncanonical NF-κB-induced gene expression changes can be determined by gene array analysis and quantitative real-time PCR.

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Author information

Authors and Affiliations

  1. Department of Microbiology and Molecular Genetics, University of Pittsburgh, School of Medicine, 1.18 Hillman Cancer Center, 5117 Centre Avenue, Pittsburgh, PA, 15213, USA

    Zhaoxia Qu & Gutian Xiao

Authors
  1. Zhaoxia Qu
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  2. Gutian Xiao
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Corresponding author

Correspondence to Gutian Xiao .

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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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Qu, Z., Xiao, G. (2015). Systematic Detection of Noncanonical NF-κB Activation. 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_8

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  • DOI: https://doi.org/10.1007/978-1-4939-2422-6_8

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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

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