Characterization of SLP-76-Grb2 and Grb2-Sos signaling complexes. Immunoprecipitation was performed with anti-Sos, anti-Cbl, and anti–SLP-76 antibody from lysates of resting U937IF cells or cells stimulated by anti-FcγRI cross-linking with MoAb 32.2 F(ab′)2 fragment. Proteins were resolved by SDS/PAGE, transferred to nitrocellulose membrane, and immunoblotted. (A) Antiphosphotyrosine immunoblot. Lane 1 represents precipitation with preimmune antisera. Lanes 2 and 3 represent anti-Sos IP of U937IF cells at rest and after 5 minutes stimulation, respectively. Lanes 4 and 5 represent anti-Cbl IP of U937IF cells at rest and after 5 minutes stimulation, respectively. Lanes 6 and 7 represent anti–SLP-76 IP of U937IF cells at rest and after 5 minutes stimulation, respectively. Lane 8 represents whole-cell lysate (1 × 106 cell equivalents) of stimulated U937IF cells. (B) Same membrane as in Fig 5A was blocked and reprobed. Upper panel represents anti-Sos immunoblot. Second panel represents anti-Cbl immunoblot. Third panel represents anti–SLP-76 immunoblot. Lower panel represents anti-Grb2 immunoblot.
