Methods to quantify the NF-κB pathway during senescence

Research output: Chapter in Book/Report/Conference proceedingChapter

14 Scopus citations


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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages20
StatePublished - 2019

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745

Bibliographical note

Publisher Copyright:
© Springer Science+Business Media, LLC, part of Springer Nature 2019.


  • Aging
  • NF-κB
  • SASP
  • Senescence
  • In Situ Hybridization/methods
  • Humans
  • Biomarkers/metabolism
  • Protein Transport
  • Animals
  • Cellular Senescence
  • Cell Nucleus/genetics
  • Immunoenzyme Techniques/methods
  • NF-kappa B/genetics

PubMed: MeSH publication types

  • Journal Article


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