Investigating mechanisms that control ubiquitin-mediated DAF-16/FOXO protein turnover

Thomas Heimbucher, Coleen T. Murphy

Research output: Chapter in Book/Report/Conference proceedingChapter


Protein turnover of FOXO family transcription factors is regulated by the ubiquitin-proteasome system. A complex interplay of factors that covalently attach certain types of ubiquitin chains (E3-ubiquitin ligases), and enzymes that are able to remove ubiquitin conjugates (deubiquitylases), regulate the degradation of FOXO proteins by the proteasome. Here, we describe methods to characterize candidate E3-ubiquitin ligases and deubiquitylases as regulators of the FOXO ubiquitylation status. Our protocol can be utilized to purify and enrich a ubiquitylated FOXO pool from cultured cells under denaturing conditions, which inactivates cellular deubiquitylases and thereby protects ubiquitin conjugates on FOXO proteins. In addition, our method describes how ubiquitylated FOXO proteins can be renatured in a stepwise fashion to serve as substrates for in vitro deubiquitylation (DUB) assays.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages9
StatePublished - 2019

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745

All Science Journal Classification (ASJC) codes

  • Genetics
  • Molecular Biology


  • DAF-16
  • Deubiquitylation (DUB) assay
  • FOXO
  • Proteasome
  • Protein stability
  • Ubiquitin
  • Ubiquitylation assay


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