Bcl-3 binds Fyn in transfected cells and activated platelets. (a) COS-7 cells, transfected with vector or Bcl-3 cDNA, were incubated alone or with agarose-conjugated fusion proteins consisting of GST, GST-Grb2, or GST-Fyn for 2 hr. After extensive washing, protein was eluted from the beads and Bcl-3 was detected by Western blot analysis. (b) Bcl-3 protein expression was detected in cellular lysates from COS-7 cells transfected with Bcl-3 cDNA and subsequently incubated with GST-Fyn fusion proteins that contained either its SH2-SH3 domains, the SH2 domain, or the SH3 domain. Binding of Bcl-3 to the GST-Fyn fusion protein containing both the SH2-SH3 domains was also examined under more stringent conditions (high salt = 400 mM, right lane). (c) Platelets were activated with thrombin (0.1 unit/ml for 2 hr), the cellular lysates were immunoprecipitated with an antibody against Fyn or mouse IgG, and Bcl-3 protein was detected by Western blot analysis as described in Materials and Methods. This figure is representative of four experiments.