Fig. 6. Wnt-5a cooperates with Wnt-3a in the phosphorylation of Dvl2, but inhibits Wnt-3a-induced activation of -catenin. (A) Increasing doses of Wnt-5a (50, 100, 300, 500, 1000 ng/ml) increase Dvl2 phosphorylation in vehicle (DMSO)-treated SN4741 cells and compete in the blockade of CK1 by D4476. (B-E) SN4741 cells were pretreated with 100 ng/ml of Wnt-5a (B,D) or 20 ng/ml of Wnt-3a (C,E) in the presence (D,E) or absence (B,C) of D4476 (100 µM) for 5 minutes. Subsequently, Wnt-3a (50, 100 and 200 ng/ml) or Wnt-5a (100, 200 and 500 ng/ml), was added for 2 hours. In A-E the phosphorylation of Dvl2 was detected by western blotting as phosphorylation-dependent mobility shift (dephosphorylated, ; phosphorylated, ). In B-E the activation of -catenin (ABC) was determined using an antibody against Ser33/37-dephosphorylated -catenin. Results shown in B-E were quantified using densitometry and either normalised to untreated control for ABC or shown as a ratio of phosphorylated:dephosphorylated for Dvl2. Duplicate experiments showed comparable results.