[Biochemical characterization of SLP-76-Cbl and SLP-76-Shc interactions.] Immunoprecipitation was performed with 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 for varying periods of time ranging from 30 seconds to 30 minutes in the presence or absence of PAP. Proteins were resolved by SDS/PAGE, transferred to nitrocellulose membrane, and immunoblotted. (A) Antiphosphotyrosine immunoblot. Lane 1 represents precipitation with preimmune antisera. Lanes 2-6 represent anti–SLP-76 IP of U937IF cells at rest and after 30 seconds, 2 min, 10 min and 30 min stimulation respectively. Lanes 7-11 represent anti-SLP-76 IP of U937IF cells at rest and after 30 seconds, 2 minutes, 10 minutes, and 30 minutes stimulation, respectively, to which 1.8 units PAP was added to the cell lysates. Lane 12 represents whole-cell lysate (1 × 10⁶ cell equivalents) of stimulated U937IF cells. (B) Same membrane as in Fig 3A was blocked and reprobed. Upper panel represents anti-Cbl immunoblot. Second panel represents anti–SLP-76 immunoblot. Third panel represents anti-Shc immunoblot. Lower panel represents anti-Grb2 immunoblot.