Fig. 1. Fz function is required for Wnt-induced LRP6 phosphorylation. (A) Shisa inhibited Wnt3a-induced LRP6 phosphorylation. HEK293T cells cotransfected with LRP6 plus Flag-tagged Shisa (or a control vector) were incubated with Wnt3a conditioned medium (CM) or the control CM for 1 hour. Shisa inhibited Wnt3a-induced β-catenin accumulation in the cytosol. β-actin: a loading control. (B) L cells stably transfected with shRNAs against Fz2 and Fz7 [L (Fz-) cell] showed diminished Wnt-induced LRP6 (endogenous) phosphorylation. The L (Fz-) cells were treated for 1hour with increasing concentrations of Wnt3a CM. These L (Fz-) cells also exhibited attenuated β-catenin stabilization in response to Wnt3a compared to the control L cells. (C) Human Fz5 expression rescued Wnt3a-induced LRP6 (endogenous) phosphorylation in the L (Fz-) cells. The stable clones of L (Fz-) cell transfected with Fz5 (or the control vector) were pooled together and assayed.