Association of Grb-2 with IRS-1 during insulin, but not IFN-α stimulation. Antiphosphotyrosine immunoblots are shown. (A) U-266 cells were incubated for 5 minutes at 37°C in the presence or absence of IFN-α or insulin as indicated. Cell lysates were immunoprecipitated with either control nonimmune rabbit Ig (RIgG) (lane 1) or a polyclonal antibody against Grb-2 (lanes 2 through 4). The identity of the 116-kD tyrosine phosphorylated protein seen in lanes 3 and 4 is unknown. (B) U-266 cells were serum-starved for 1 hour and were subsequently incubated for 5 minutes at 37°C in the absence (lane 1) or presence of IFN-α (lane 2) or insulin (lane 3). Cell lysates were bound to a GST fusion protein containing the SH2 domain of Grb-2, before SDS-PAGE analysis and immunoblotting. (C) U-266 cells were serum starved for 2 hours and were subsequently either not treated (lane 1) or treated with IFN-α (lanes 2 and 4) or insulin (lanes 3 and 5) for 5 minutes. Cell lysates were bound to either a GST fusion protein containing the nSH2 domain of p85 or to GST alone as indicated, before SDS-PAGE analysis and immunoblotting. (D) U-266 cells were serum starved for 1 hour and were subsequently either not treated (lane 3) or treated with IFN-α (lanes 1 and 4) or insulin (lanes 2 and 5) for 5 minutes. Cell lysates were bound to either a GST fusion protein containing the cSH2 domain of p85 or to GST alone as indicated, before SDS-PAGE analysis and immunoblotting.