A Simple Method to Assess Abundance of the ß-Catenin Signaling Pool in Cells.

ß-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 ß-catenin (e.g., TCF-dependent reporter assays, nuclear accumulation of ß-catenin, and generation of N-terminally hypophosphorylated ß-catenin), there are cell-type and context-dependent limitations of these methods. Since the posttranscriptional stabilization of ß-catenin outside of the cadherin complex appears universally required for ß-catenin signaling, the following method allows for simple assessment of the cadherin-free fraction of ß-catenin in cells, using a GST-tagged form of ICAT (Inhibitor of ß-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.