β-Cat LOF and GOF in MBEs in vitro. β-Cat^lox/lox and β-cat^{lox(ex3)/lox(ex3)} MBEs were treated with TAT-Cre in vitro to achieve β-cat LOF and GOF, respectively. (A, a–c and f–h) β-Cat^lox/lox with or without TAT-Cre, stimulated with controlCM (CCM) or Wnt3aCM. IF staining reveals increased junctional Cldn3 in Cre⁻/Wnt3aCM and a complete absence of β-cat in Cre⁺/Wnt3aCM. (d, e, i, and j) β-Cat^{lox(ex3)/lox(ex3)} MBEs with or without TAT-Cre, and IF stained for Cldn3 and β-cat. Arrowheads point to nuclear β-cat. (B) Quantitative RT-PCR for Cldn3 in Cre⁻/Wnt3aCM-treated cells from β-cat^lox/lox (n = 3; **, P = 0.0048) and in Cre⁺/controlCM cells from β-cat^{lox(ex3)/lox(ex3)} (n = 3; *, P = 0.0341) mice. Other tested genes were not significantly altered. Axin2 induction confirmed canonical Wnt signaling in Cre⁻/Wnt3aCM-treated cells from β-cat^lox/lox (n = 3; **, P = 0.0006) and in Cre⁺/controlCM cells from β-cat^{lox(ex3)/lox(ex3)} (n = 3; **, P = 0.0029) mice. Controls are set as 100% (dashed lines). Error bars represent SEM. (C) Western blot analysis for Cldn3 in β-cat^lox/lox and β-cat^{lox(ex3)/lox(ex3)} cells. Transcription and translation of an N-terminal truncated form of β-cat are detected by the anti–β-cat antibody and represented by the bottom band in β-cat^{lox(ex3)/lox(ex3)}/Cre⁺ cells. See graphs for densiomeric quantification of Western blot bands. Data are representative of at least three independent experiments.