Figure 1: Daple regulates the activation of Rac in the non-canonical Wnt signalling pathway.

(a) Proposed domain structure of mouse Daple. Daple can be divided into three domains, an N-terminal (NT) domain, a central coiled-coil domain and a CT domain. An arrow indicates the portion of Daple used as an antigen for the production of polyclonal anti-Daple antibody. (b) Endogenous expression of Daple in HEK293T cells shown by western blot analysis (IB) using anti-Daple antibody. The band at 250 kDa detected with anti-Daple antibody corresponded to that detected by anti-myc antibody in myc epitope-tagged Daple (Daple-myc)-transfected cells. (c, d) The activities of Rac, Cdc42 and Rho in HEK293T cell lysates transfected with either control (empty) or Daple-myc were assessed by GST-PAK-PBD or GST-Rhotekin RBD pull-down assays. The precipitates and total cell lysates (TCL) were analysed with the indicated antibodies (c). The intensity of each band for Rac, Cdc42 and Rho was quantified, and the results are presented as fold-induction of the activity of the small GTPases by Daple-myc, compared with the activities in control cells that are expressed as 1.0 (d). (e, f) Activity of Rac, Cdc42 and Rho in HEK293T cell lysates transfected with either control or Daple siRNA. The precipitates and TCL were analysed with the indicated antibodies (e) and quantified (f). (g, h) HEK293T cells transfected with either control or Daple siRNA were stimulated with recombinant Wnt5a (100 ng ml⁻¹) for 1 h. Rac activation was monitored by GST pull-down assays (g). The intensity of each active Rac band was quantified, and the results are presented as the fold-induction of the activity of Rac by Wnt5a, compared with the levels in the absence of Wnt5a that are defined as 1 (h). All bars represent the mean values ±s.d. of three experiments. Significant difference was determined by Student's t-test. *P<0.05, as compared with control cells; n.s., not significant.