FIG. 7.

Dkk1 inhibits the Wnt/β-catenin pathway but does not block Dvl phosphorylation. (A) Dkk1 does not inhibit endogenous Dvl phosphorylation. 293T cells were transfected with vectors encoding either Dkk1 or Fzd8 Ex, and Dvl phosphorylation was analyzed by Western blotting. Expression of epitope-tagged Dkk1 and Fzd8 Ex was confirmed using anti-V5 and anti-Myc antibodies, respectively. (B) Dkk1 inhibits TCF/β-catenin-dependent transcription. 293T cells were transfected with vectors encoding Dkk1 or Fzd8 Ex, together with pTOPflash and pRL-TK. These cells were then cocultured with Rat2 cells stably expressing either Wnt1 (black bars) or empty vector (white bars). Luciferase assays were performed as described for Fig. 5C. Results shown are the mean + standard deviation of six replicates. (C) Dkk1 inhibits Wnt1-dependent β-catenin stabilization. 293T cells were transfected either with empty vector or vector encoding Wnt1, in the absence or presence of vectors encoding Dkk1 or Fzd8 Ex. Cytosolic β-catenin was analyzed by Western blotting as described before, and GSK3β was used as a loading control. (D) Dkk1 inhibits Wnt3a-mediated β-catenin stabilization but not Dvl phosphorylation. 10T1/2 cells were pretreated for 2 h with either control or Dkk1 CM, and this was then supplemented with control or Wnt3a CM for a further 2 h. Cell extracts were analyzed for β-catenin levels and Dvl phosphorylation as described above.