Fig. 3. Dvl is required for Wnt-induced LRP6 phosphorylation and acts via the DIX and PDZ domains. (A) Reduction of Dvl proteins diminished Wnt3a-induced Lrp6 (endogenous) phosphorylation. MEFs lacking Dvl1 and Dvl2 (Dvl1^-/-;Dvl2^-/-) (see Fig. S1 in the supplementary material) were infected with each of the four different lentiviral shRNAs against mouse Dvl3. After 3 days, the cells were treated with Wnt3a CM or control CM for 1 hour. Dvl3 protein level were drastically reduced by shRNA2, 3 or 4. Dvl3 protein exhibited typical Wnt-induced mobility shift (due to phosphorylation). shRNA2 and 4 were used in further experiments and yielded identical results. (B) The wild-type Dvl2 rescued LRP6 (endogenous) phosphorylation in Dvl3 knockdown Dvl1^-/-;Dvl2^-/- MEFs. Dvl1^-/-;Dvl2^-/- MEFs stably expressing Dvl2 (lanes 5 to 8) or the control vector (lanes 1 to 4) were pooled and infected with the lentiviral Dvl3 shRNA as indicated, then treated with Wnt3a or control CM for 1 hour as indicated. The exogenously expressed Dvl2 was detected by the Flag tag. (C) Dvl2DEP, but neither Dvl2DIX nor Dvl2PDZ, rescued the Wnt3a-induced Lrp6 (endogenous) phosphorylation in Dvl3 knockdown Dvl1^-/-;Dvl2^-/- MEFs. Dvl1^-/-;Dvl2^-/- MEFs stably expressing the control vector (lanes 1-4), Dvl2DIX (lanes 9-12), Dvl2PDZ (lanes 5-8) or Dvl2DEP (lanes 13-16) were infected with the lentiviral Dvl3 shRNA as indicated, and treated with Wnt3a CM or control CM for 1 hour as indicated. The expression of Dvl2 mutants was detected by the Flag tag.