Binding of 3BP2 to Vav1 in B cells. (A) Interaction between endogenous 3BP2 and Vav1 in B cells. Daudi cells were activated for 0 or 5 minutes with anti-IgM antibody and lysed in NP-40 lysis buffer supplemented with 0.1% SDS. Immunoprecipitates were prepared with anti-3BP2 or irrelevant (control) antibodies and probed with antiphosphotyrosine antibody. The membranes were then stripped and reprobed with antibodies against 3BP2 and Vav1. (B) Interaction between 3BP2 and Vav1 in lipid raft-dissociating conditions. Unstimulated Daudi cells were lysed in 1% octylglucoside lysis buffer, and immunoprecipitates were prepared with anti-Vav1 or irrelevant (control) antibodies and probed with anti-3BP2 antibody. The membranes were then stripped and reprobed with antibodies to Vav1. (C-D) In vitro interaction between 3BP2 and Vav proteins. Lysates from unstimulated (–) or anti-IgM–stimulated (+) Daudi cells were mixed with GST or GST Vav2 SH3-SH2-SH3 fusion proteins (B), or with GST or GST 3BP2 SH2 fusion proteins (C) that were immobilized on glutathione beads. Bound proteins were detected by immunoblotting with the indicated antibodies.