Grb2 associates through its SH2 domain with Shc and through its SH3 domain with p140, and both interactions are hGM-CSF–dependent. (A) Binding of Grb2 to tyrosine-phosphorylated Shc is mediated through the SH2 domain of Grb2. Cell lysates from unstimulated (lanes 1, 3, and 5) or hGM-CSF–stimulated (lanes 2, 4, and 6) TF-1 cells were immunoprecipitated (IP) with anti-Shc (lanes 1 and 2) or anti-Grb2 (lanes 3 and 4) antibodies or adsorbed with a GST fusion protein containing the SH2 domain of Grb2 (Grb2 SH2; lanes 5 and 6). The precipitates were analyzed by SDS-PAGE followed by antiphosphotyrosine immunoblotting (lanes 1 through 6). Arrows indicate the position of the detected phosphotyrosyl proteins and the GST-Grb2-SH2 fusion protein. (B) Binding of Grb2 to tyrosine-phosphorylated p140 is mediated through the SH3 domains of Grb2. Cell lysates of unstimulated (lanes 1, 3, 5, and 7) or hGM-CSF–stimulated (lanes 2, 4, 6, and 8) TF-1 cells were immunoprecipitated with anti-Shc antibodies (lanes 1 and 2) or adsorbed with either a GST fusion protein containing the C-terminal SH3 domain of Grb2 (GST Grb2 SH3-C; lanes 3 and 4), the N-terminal SH3 domain of Grb2 (GST Grb2 SH3-N; lanes 5 and 6), or with GST vector protein (GST) (lanes 7 and 8). The precipitates were analyzed by antiphosphotyrosine immunoblotting (lanes 1 through 8).