A Simple Method to Assess Abundance of the β-Catenin Signaling Pool in Cells
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 (Inhibitor of β-Catenin and Tcf) as an affinity matrix. This method is more sensitive and quantitative than immunofluorescence and may be useful in studies that implicate TCF-independent signaling events.
Key wordsWnt β-catenin ICAT TCF (T-cell factor) GST (glutathione S transferase) Fusion proteins Affinity precipitation Western blot
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