Activation of Wnt signaling by Fam13a. (A) Morphology of uninjected and Fam13a RNA (1 ng)–injected Xenopus embryos at the tadpole stage. Embryos injected with Fam13a developed partial secondary axes (arrowheads). (B) Expression of siamois, Xnr3, chordin, noggin, sizzled, sox17, and Xbra in control and Fam13a-injected embryos. Embryos were harvested at the early gastrula stage. (C) Epistasis analysis showing that the expression of siamois and Xnr3 in Fam13a-overexpressed animal caps was blocked by coexpression of axin or GSK3ß. (D) Western blot showing that overexpression of Fam13a increased the expression of myc–β-catenin in Xenopus embryos. At the two-cell stage, embryos were injected with a mixture of myc–β-catenin (100 pg) and myc-GFP (50 pg) RNAs or a mixture of myc–β-catenin, myc-GFP, and myc-Fam13a (1 ng). Embryos were harvested at stage 9. (E) RT-PCR, showing the expression of siamois and Xnr3 in Fam13a-, ∆315–329–, or RR340;531AA-overexpressed animal caps. As a control, the levels of injected Fam13a, ∆315-329, and RR340;531AA RNAs were monitored. (F) Western blot, showing that overexpression of Fam13a increased the expression of myc–β-catenin in A549 cells. (G) Dual-luciferase assays, showing that overexpression of Fam13a activated TOPFlash and enhanced the activity of β-catenin in A549 cells. FOPFlash, which contains mutations in the TCF binding sites, was included as a negative control. Firefly luciferase activity was normalized to the activity of Renilla luciferase (TK-RL). (H) Overexpression of Fam13a and ∆315–329, but not RR340;531, enhanced the activity of β-catenin in the TOPFlash luciferase assay in A549 cells. Firefly luciferase activity was normalized to the activity of Renilla luciferase. (I) Western blot, showing coimmunoprecipitation of Flag-Fam13a and myc-axin. Of note, we did not detect any interaction between Flag-Fam13a and myc-GSK3β. (J) Western blot showing that overexpression of Fam13a reduced the expression of HA-Axin in A549 cells. The expression level of myc-GSK3ß was not altered by Fam13a overexpression.