Figure 3: Daple regulates the interaction between Dvl and PKCλ in the activation of Rac.

(a, b) Lysates from HEK293T cells transfected with control (empty), Daple CT or Daple CTΔGCV were immunoprecipitated with either anti-Dvl2 (a) or anti-PKCλ (b) antibody. The precipitates and the total cell lysates (TCL) were analysed by western blot, using the indicated antibodies. (c) Lysates from HEK293T cells transfected with either control or Daple siRNA were immunoprecipitated with either anti-PKCλ antibody or normal mouse IgG. The precipitates and the TCL were analysed by western blot using the indicated antibodies. (d) Monolayers of confluent HEK293T cells that had been transfected with either control or Daple siRNA were serum-starved for 24 h and stimulated with Wnt5a (100 ng ml⁻¹). The monolayers were wounded and incubated for an additional 30 min. TCL were immunoprecipitated with Dvl2 antibody, followed by western blot analysis using the indicated antibodies. (e) Lysates from HEK293T cells transfected with myc-PKCλ, and either GST, GST-Daple CT, or GST-Daple CTΔGCV were precipitated with glutathione-Sepharose beads. The precipitates and the TCL were analysed by western blot, using the indicated antibodies. (f) HEK293T cells were transfected with either control plasmid or Daple CT, and were incubated in the absence or presence of the aPKC inhibitor, cell-permeable myristoylated aPKC pseudosubstrate, (20 μM) for 1 h. Rac activation was monitored by GST-PAK-PBD pull-down assays. The precipitates and the TCL were analysed by western blot, using the indicated antibodies. (g) HEK293T cells were serum-starved and incubated with Wnt5a either in the absence or presence of the aPKC inhibitor, followed by GST-PAK-PBD pull-down assays to monitor Rac activation. (h) TCL prepared from HEK293T cells transfected with control vector or Daple CT were analysed by western blot, using the indicated antibodies.