Figure 1.

Tiam1 forms a complex with ephrin-B1 and EphA2 receptor. (A) COS1 cells were transiently transfected with a plasmid encoding wild-type Tiam1 together with that encoding ephrin-B1 (lanes 1, 2, 5, 6) or EphA2 (lanes 3, 4, 7, 8). Cells were lysed and immunoprecipitated (IP) with anti-ephrin-B1 (goat polyclonal, lane 1), normal goat serum (NGS, lane 2), anti-EphA2 (lane 3), mouse IgG1 (lane 4), anti-Tiam1 (lanes 5, 7) or normal rabbit serum (NRS, lanes 6, 8). The precipitates were subjected to immunoblotting (IB) with the indicated antibodies. The same membranes were reblotted with the antibodies indicated (bottom panels). The expression of Tiam1, ephrin-B1 and EphA2 in the cell lysate was confirmed by immunoblotting. When wild-type ephrin-B1 is overexpressed in COS1 cells, at least two major bands were observed by anti-ephrin-B1 goat polyclonal antibody. Ig, immunoglobulin. The prominent band of 80 kDa in lane 3 is an unknown protein, which associates with EphA2 and may have a related structure with Tiam1. (B) Schematic representation of a wild-type and the truncated Tiam1 cDNA constructs used in this study. Proteins are depicted to scale; M, myristoylation signal; PHn and PHc, NH2- and COOH-terminal pleckstrin homology domains; TSS, otherwise known as coiled-coil region and an additional adjacent region (CC-Ex); PDZ, PSD-95/DlgA/ZO-1 domain; DH, Dbl homology domain. (C) COS1 cells were transiently transfected with the plasmids as indicated in the above lanes. The cell lysates were immunoprecipitated with anti-ephrin-B1 C18 (lanes 1, 3), EphA2 (lanes 5, 7), normal rabbit serum (lanes 2, 4) or mouse IgG1 (lanes 6, 8) and immunoblotted with anti-myc antibody. The same membranes were reblotted with anti-ephrin-B1 (goat) or anti-EphA2 as indicated. The expression of myc-tagged Tiam1 constructs in these cell lysates (total lysate) was confirmed by immunoblotting. The additional band at 110 kDa in lane 1 may be an artificially produced fragment of overexpressed Tiam1 construct.