Fig 2.

The N-terminal part of Dvl is required to interact with Rac1. (A) Deletion mapping of the Dvl3 domain mediating the interaction with Rac1 in HEK293A cells. The FLAG-Dvl3 expression constructs used in coimmunoprecipitation experiments are showed in panel B (total cell lysates [TCL] shown to the right). Note that FLAG-Dvl3 pulldown after Rac1 immunoprecipitation (IP) was lost in Dvl3 mutants lacking the N-terminal part, including the DIX domain. Arrows point to the Dvl3-FLAG variants coimmunoprecipitating with endogenous Rac1. (B) Schematic representation of the constructs used in panel A and the results obtained. + or − symbols indicate the efficiency of coimmunoprecipitation for the different mutants. (C) HEK293A cells were transfected with the indicated expression constructs of Dvl2 harboring point mutations in the DIX domain. Subcellular localization of individual Dvl2 mutants has been determined by immunocytochemical staining using anti-HA antibody. (D) Coimmunoprecipitation of all Dvl2 constructs was detected after Rac1 immunoprecipitation but not if control IgG was used instead of an anti-Rac1 antibody. (E) HEK293A cells were transfected with the indicated Dvl constructs and Rac1-MYC. Rac1 activity was determined by the level of Rac1-GTP pulldown. Note that Rac1 was not activated by the Dvl3 ΔN-FLAG construct. (F) Summary of experiments shown in panel E. *, P < 0.05; **, P < 0.01 by Student t test (compared to mock).