Fig. 3. Physical interactions between Fra, Abl and Trio. (A) The cytoplasmic domain of Fra interacts directly with Abl and Trio. Beads only (lane 1), GST bound to beads (lane 2) or GST-Fra_CYTO bound to beads (lane 3) were incubated with in vitro translated, epitope-tagged Abl or Trio. Approximately 20% of each pulldown (2 µg of fusion protein) and bound target proteins were resolved by SDS-PAGE. Bound proteins (top two blots) were visualized by anti-Myc immunoblotting, and fusion proteins (bottom gel) were visualized by Coomassie staining. `In' represents 2% of total input protein incubated with beads, GST or GST-Fra<sub>CYTO</sub>. Abl and Trio specifically interact with GST-Fra<sub>CYTO</sub> (lane 3), but not GST or beads (lanes 1 and 2). Trio<sup>SPR</sup>-Myc is deleted for the spectrin-like repeats to optimize expression in vitro. (B,C) Trio and Abl interact directly via their SH3 domains. (B) GST-TrioSH3 (lane 2), but not GST (lane 1), specifically pulls down in vitro translated Abl-Myc. (C) GST-AblSH3 (lane 2), but not GST (lane 1), specifically pulls down in vitro translated Trio<sup>SPR</sup>-Myc. `In' represents 2.5% of input incubated with GST or fusion protein. Target proteins were visualized by anti-Myc staining, whereas fusion proteins (bottom gel) were visualized by anti-GST staining. Panels B and C were assembled from different lanes on the same gel. (D,E) Fra complexes via its cytoplasmic domain with Abl and Trio in S2 cells. (D) HA-tagged Fra (arrow, lanes 1 and 2, bottom gel) or Fra^CYTO, a Fra molecule lacking the intracellular domain (arrowhead, lane 3, bottom gel), were co-expressed in S2 cells with Abl-Myc (lanes 2 and 3, middle gel), and complexes were immunoprecipitated with anti-Myc antibody. Fra-HA (arrow, top gel) co-immunoprecipitates only in the presence of Abl-Myc (compare lanes 1 and 2). Fra^CYTO-HA (arrowhead indicates the absence of Fra^CYTO-HA, top gel) does not co-immunoprecipitate with Abl-Myc (compare lanes 2 and 3). When expressed in S2 cells, Abl-Myc runs as a 180-190 kDa doublet, similar to untagged Abl (D, compare with Fig. 4C,D). Additional low-mobility bands in D (top gel, lanes 2 and 3) are background staining of Abl-Myc, which is nearly the same size as Fra-HA. (E) Fra-HA (lane 2) but not Fra^CYTO-HA (lane 3) co-immunoprecipitates with Trio-Myc.