Ectopic expression of γ-cateninS28A in mESCs activates the Wnt pathway without altering cytosolic β-catenin levels.

<p>(A) mESCs were stably transfected with a Myc-tagged S28A variant of γ-catenin. Clones were isolated, expanded and screened for γ-catS28A expression by western blotting of hypotonic lysates using a tag-specific antibody. (B) Alkaline phosphatase staining of WT, γ-catS28A-expressing and β-catS33A-expressing mESC colonies, visualized using bright-field microscopy. Bar = 400 µm. (C) Western blot analyses of nuclear and cytosolic extracts prepared from WT and γ-catS28A cell lines, as indicated. Elevated levels of γ-cat protein were detected in both cytoplasmic and nuclear lysates of γ-catS28A-overexpressing cell lines. (D) TCF reporter activity of mESCs stably expressing γ-catS28A. (E) Induction of the β-catenin/TCF target genes, <i>Axin2, Brachyury</i> and <i>Cdx1</i>, but not <i>c-Myc</i>, in mESCs stably expressing γ-catS28A or β-catS33A. (F) Expression of stabilized γ-catenin in WT mESCs does not result in detectable levels of cytosolic β-catenin as determined by western blot analysis of hypotonic lysates. Cytosolic levels of β-catenin in GSK-3 DKO mESCs or WT mESCs treated with CHIR99021 (24 h, 15 µM) are provided as positive controls. For (D) and (E), Bars represent means and error bars indicate s.e.m. (n = 3).