Fig. 4. Nuclear β-catenin signaling in TS cells in culture. Western blotting analysis of Wnt-family components in TS cells. (A) Time-dependent accumulation and nuclear translocation of active β-catenin in response to recombinant Wnt3a protein (50 ng/ml) in differentiating TS cells. (B) Wnt3a-induced c-Myc and Ppar expression in differentiating TS cells. (C) Dkk1 (1 µg/ml) and PKF115-584 (1 µM) blocked Wnt3a-induced cytoplasmic accumulation of active dephosphorylated β-catenin in TS cells by 2 hours of co-treatments. Notably, even basal levels of nuclear β-catenin disappeared following PKF115-584 treatment. (D) Similar treatment of PKF115-584 (1 µM) downregulated Wnt3a-induced c-Myc and Ppar expression in differentiating TS cells. (E) Dvl1-3 proteins in TS cells. Whereas Dvl1 was only detected in the nucleus, Dvl2 and Dvl 3 were detected in both the cytoplasm and nucleus in response to Wnt3a. (F) Wnt receptors Fzd2, Fzd4, Lrp5, Lrp6, Kremen1 and Kremen2 in TS cells. Wnt3a facilitated nuclear import of Wnt receptor subtypes in differentiating TS cells, mimicking the finding in blastocysts during activation. C, M and N indicate cytoplasmic, membrane and nuclear protein extraction, respectively.