Fig. 5. Palladin specifically interacts with Eps8. (A) Schematic representation of the palladin protein used as bait. The amino acid positions are indicated by the flanking numbers. The structural domains are shown as boxes: the N-terminal half of the molecule contains a serine-rich (S) and a proline-rich (P) domain, and the C-terminal half of the molecule contains three tandem repeats of a domain called `IgC2'. (B) Schematic representation of full-length Eps8 and the truncated form found in the yeast two-hybrid screen. The amino acid positions are indicated as in A. The structural domains are shown as boxes: a phosphotyrosine-binding domain (PTB), an SH3 domain and an effector region (ER). (C) Palladin and Eps8 form complexes in cultures cells. Left panel: alladin was immunoprecipitated with 1E6 monoclonal antibody from lysed Swiss 3T3 fibroblasts, and the samples were probed with anti-Eps8 monoclonal antibodies to determine whether Eps8 co-precipitates with palladin. Whole cell lysate was run as positive control, and a sample that contained no primary antibody (beads alone) was run as negative control. Right panel: Eps8 was immunoprecipitated with anti-Eps8 antibody and blotted for palladin, also with whole cell lysate and beads alone as positive and negative controls, respectively. Note that Eps8 was detected in the palladin immunoprecipitate and palladin was detected in the Eps8 immunoprecipitate. L, whole cell lysate; C, beads alone; Ab, antibody used for IP. (D) Western blot analysis from cytosolic extracts of A7r5 cells treated with PDGF. Cells were untreated (SS, serum starvation) or treated with PDGF for 5, 10 and 30 minutes (5', 10' and 30'). Blots were probed with anti-palladin 1E6 monoclonal antibody. (E) Co-immunoprecipitation of palladin and Eps8 from cytosolic extracts of Eps8 null mouse embryonic fibroblasts (MEF Eps8^-/-) and rescued MEF (MEF Eps8^-/- + myc-Eps8). Cells were untreated (SS) or treated with PDGF for 10 and 30 minutes (10' and 30'). Eps8 antibody was used for immunoprecipitation. Blots were probed with anti-Eps8 Abs or anti-palladin 1E6 monoclonal antibody.