A simple method to assess abundance of the β-Catenin signaling pool in cells

Annette S. Flozak, Anna P. Lam, Cara J. Gottardi

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

β-catenin (CTNNB1) is a dual-function cell–cell adhesion/transcriptional co-activator protein and an essential transducer of canonical Wnt signals. Although a number of established techniques and reagents are available to quantify the nuclear signaling activity of β-catenin (e.g., TCF-dependent reporter assays, nuclear accumulation of β-catenin, and generation of N-terminally hypophosphorylated β-catenin), there are cell-type and context-dependent limitations of these methods. Since the posttranscriptional stabilization of β-catenin outside of the cadherin complex appears universally required for β-catenin signaling, the following method allows for simple assessment of the cadherin-free fraction of β-catenin in cells, using a GST-tagged form of ICAT (I nhibitor of β- Ca tenin and T cf) as an affi nity matrix. This method is more sensitive and quantitative than immunofl uorescence and may be useful in studies that implicate TCFindependent signaling events.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages49-60
Number of pages12
DOIs
StatePublished - 2016

Publication series

NameMethods in Molecular Biology
Volume1481
ISSN (Print)1064-3745

Keywords

  • Affi nity precipitation
  • Fusion proteins
  • GST (glutathione S transferase)
  • ICAT
  • TCF (T-cell factor)
  • Western blot
  • Wnt
  • β-catenin

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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