Fig. 5. Axin is required for Lrp6 phosphorylation via its ability to bind Gsk3, and inhibition of Gsk3 at the plasma membrane blocks Wnt/β-catenin signaling. (A) Axin^-/- and the wild-type ES cells were treated with Wnt3a or control CM. Wnt3a-induced Lrp6 (endogenous) phosphorylation was significantly reduced in Axin^-/- ES cells. (B,C) Reducing Axin2 expression in Axin^-/- ES cells further inhibited Lrp6 (endogenous) phosphorylation (B). Axin^-/- ES cells were infected with lentiviral shRNAs against mouse Axin2. TfR, transferin receptor, loading control. The efficiency of Axin2 mRNA knockdown was assessed by RT-PCR analysis. GAPDH, a loading control (C). (D) The wild-type axin, but not the GSK3 binding mutant axin (L396Q) promoted LRP6 phosphorylation by GSK3. Axin and axin (L396Q) were cotransfected with VSVG-tagged LRP6 in the presence or absence of GSK3 in HEK293T cells. Axin and GSK3 were detected by the Flag and HA tags, respectively. (E) Gsk3 and Gsk3β share redundant function in Wnt-induced LRP6 phosphorylation. ES cells null for both Gsk3 and Gsk3β (Gsk3^-/-;β^-/-), null for either Gsk3 (Gsk3^-/-) or Gsk3β (Gsk3β^-/-) and the control wild-type ES cells were treated with Wnt3a or control CM for 1 hour. The endogenous Lrp6 was examined. β-actin, loading control. (F) A plasma membrane-targeted CAAX-GID blocked wnt8 (Xwnt8) signaling in Xenopus embryo explants. GID induced nr3 (Xnr3) expression; CAAX-GID, but not CAAX-GID-LP, blocked wnt8 (Xwnt8)-induced nr3 expression (each was injected at 10-50 pg mRNA/embryo). wnt8 was injected at 10 pg mRNA/embryo. -, without reverse transcriptase; Un, uninjected control embryo; EF-1; loading control.