Fig. 1. Profilin binds to the FH1 domain of Daam1. (A) Schematic diagram of the Daam1 and Profilin1 constructs showing domains, with numbers indicating amino acid positions. (B,C) Coimmunoprecipitation experiments. Plasmids encoding tagged-Daam1 or Daam1 fragments and Profilin1 were cotransfected into HEK293T cells, and lysates were immunoprecipitated (IP) with indicated Abs. Precipitates were immunoblotted with indicated Abs. (B) Profilin1 interacts with Daam1, C-Daam1 and the FH1 domain of Daam1 but not N-Daam1 or the FH2 domain of Daam1. Note the tagged FH1 domain migrates overlapping the IgG band and is not resolved and a nonspecific band comigrates at the same position as HA-Daam1 (arrowhead). (C) Endogenous Profilin1 is precipitated with epitope tagged-Daam1, C-Daam1 or the FH1 domain of Daam1 but not N-Daam1 or the FH2 domain of Daam1. (D) Profilin1 does not induce Rho activation or interfere with Rho activation induced by C-Daam1. Immunoprecipitation and Rho activation assays were carried out as described (Habas et al., 2003; Habas et al., 2001).