[The Grb2-binding domain of SLP-76 does not bind Cbl.] U937IF cells at rest and stimulated by anti-FcγRI cross-linking with MoAb 197 were incubated with SLP-76 GST-fusion proteins representing the Grb2-binding domain (amino acid residues 225-265) and the proline-rich region that does not associate with Grb2 (amino acid residues 268-416). Lane 1 represents precipitation with preimmune antisera. Lanes 2 and 3 represent incubation of GST with U937IF cells at rest and after 5 minute stimulation, respectively. Lanes 4 and 5 represent incubation of SLP-76 GST-fusion protein aa 225-268 with U937IF cells at rest and after 5 minute stimulation, respectively. Lanes 6 and 7 represent incubation of SLP-76 GST-fusion protein aa 268-416 with U937IF cells at rest and after 5 minutes stimulation, respectively. Lane 8 represents whole-cell lysate (1 × 10⁶ cell equivalents) of stimulated U937IF cells. Upper panel represents anti-Cbl immunoblot. Middle panel represents anti-Shc immunoblot. Lower panel represents anti-Grb2 immunoblot.