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Cellular Senescence pp 231–250Cite as

Methods to Quantify the NF-κB Pathway During Senescence

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1896))

Abstract

Nuclear factor κB (NF-κB) is a family of transcription factors important for regulating innate and adaptive immunity, cellular proliferation, apoptosis and senescence. The NF-κB family is comprised of five subunits, RelA/p65, RelB, C-Rel, p50 (p105/NF-κB1), and p52 (p100/NF-κB2). NF-κB activity goes up with age in multiple tissues. The two subunits RelA/p65 and p50 have been implicated in senescence and aging with genetic deletion of p65 and p50 reducing or increasing senescence respectively. Pharmacologic inhibition of NF-κB also extends health span and reduces senescence in mouse models of accelerated aging. In addition, NF-κB regulates expression of many of senescence associated secretory phenotype (SASP) factors released by certain types of senescent cells that drives loss of tissue homeostasis and secondary senescence. To measure NF-κB activity with aging in vivo, multiple methods can and need to be utilized including cellular localization of p65, EMSA analysis of NF-κB DNA binding, RNA in situ hybridization, and analysis of expression of NF-κB target genes. To colocalize NF-κB activation and senescence, p65 localization or transcriptional activity can be measured by immunostaining or RNA in situ hybridization for NF-κB regulated genes along with methods such as immunostaining for γH2AX or RNA in situ for senescence markers like p16INK4a and p21. These and related methods will be described in this chapter.

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Author information

Author notes

  1. Paul D. Robbins

    Present address: Department of Biochemistry, Molecular Biology and Biophysics, Institute on the Biology of Aging and Metabolism, University of Minnesota, Minneapolis, MN, USA

Authors and Affiliations

  1. Department of Molecular Medicine and Center on Aging, The Scripps Research Institute, Jupiter, FL, USA

    Lei Zhang, Jing Zhao, Aditi Gurkar, Laura J. Niedernhofer & Paul D. Robbins

  2. Disease Biology and Cellular Pharmacology, Recursion Pharmaceuticals, Salt Lake, UT, USA

    Jing Zhao

  3. Aging Institute, Division of Geriatric Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA

    Aditi Gurkar

Authors
  1. Lei Zhang
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  2. Jing Zhao
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  3. Aditi Gurkar
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  4. Laura J. Niedernhofer
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  5. Paul D. Robbins
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Corresponding author

Correspondence to Paul D. Robbins .

Editor information

Editors and Affiliations

  1. ERIBA, University Medical Center Groningen, Groningen, Groningen, The Netherlands

    Marco Demaria

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Zhang, L., Zhao, J., Gurkar, A., Niedernhofer, L.J., Robbins, P.D. (2019). Methods to Quantify the NF-κB Pathway During Senescence. In: Demaria, M. (eds) Cellular Senescence. Methods in Molecular Biology, vol 1896. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8931-7_18

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  • DOI: https://doi.org/10.1007/978-1-4939-8931-7_18

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8930-0

  • Online ISBN: 978-1-4939-8931-7

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