The activated EPO-R binds directly to the SH2 domains of Grb2 and Shp2. CTLL-EPO-R cells were depleted of cytokine for 8 hours and stimulated with no factor (lanes 1 through 4, and 13), 50 U of murine IL-2 per milliliter (lanes 5 through 8, and 14) or 50 U of human EPO per milliliter (lanes 9 through 12, and 15) for 10 minutes. Lysates were incubated with 10 μg of GST (lanes 1, 5, and 9), GST-SH2-Grb2 (lanes 2, 6, and 10), GST-(N + C)-SH2-Shp2 (lanes 3, 7, and 11) or GST-SH2-Shc (lanes 4, 8, and 12). The immunoblot was probed with 4G10 monoclonal antiphosphotyrosine antibody followed by HRP-sheep anti-mouse IgG. A diffuse, constitutively phosphorylated 60-kD phosphoprotein associates with GST-(N + C)-Shp2 (lanes 3, 7, and 11). Lysate controls from CTLL-EPO-R cells are illustrated in lanes 13 through 15. The migration of EPO-R, Shp2, and Shc were determined by stripping and reprobing the membrane as shown in Fig 4 (data not shown). Molecular mass standards are indicated. Ab, antibody.