Fig 3.

Tiam1 is a novel Dvl binding partner. (A) Tiam1 is present in Dvl3-associated complexes, identified by LC-MS/MS after endogenous Dvl3 coimmunoprecipitation. (B) Tiam1-FLAG protein (red) localizes both in the cytosol and at the membrane (arrow) upon overexpression in SN4741. Topro3 was used to counterstain the cell nucleus. (C) Coexpression of Dvl2-EGFP (green) and Tiam1-FLAG (red) in SN4741 cells leads to a cytoplasmic colocalization of Dvl2-EGFP and Tiam1-FLAG in puncta. Arrowheads show that a small pool of Tiam1-FLAG that did not colocalize with Dvl2-EGFP remained in the membrane. (D) Dvl2-HA was found to form a complex with Tiam1-FLAG, as assessed by immunoprecipitation with Tiam1-FLAG in HEK293A cells (first panel, last lane). The next panel shows total cell lysates (TCL). (E) Dvl3-FLAG was also immunoprecipitated by Tiam1-FLAG in HEK293A cells. The complex formed by Tiam1-FLAG and Dvl3-FLAG was detected by immunoprecipitation with either anti-Dvl3 or anti-Tiam1 antibodies. (F and G) Endogenous Dvl3 (F) or endogenous Dvl2 (G) were immunoprecipitated from whole adult mouse brain lysates, and the presence of Tiam1 has been detected with an anti-Tiam1 antibody. IgG, control antibody.