Tyrosine phosphorylation of Shc proteins in hGM-CSF–stimulated TF-1 cells and identification of an associated 140-kD phosphotyrosyl protein. (A) Starved TF-1 cells were stimulated for the indicated times with 25 ng/mL hGM-CSF or with the indicated concentrations of hGM-CSF for 5 minutes at 37°C. Cells were lysed and aliquots containing 1 mg protein were immunoprecipitated with anti-Shc antibodies. Immunoprecipitates were subjected to Western blot analysis with antiphosphotyrosine antibodies. The positions of the Shc proteins (p46, p52, and p66) and p140 are indicated. The apparent molecular weights of the Shc proteins p46^Shc, p52^Shc, and p66^Shc in our gel system were consistently 51, 56, and 68 kD. To avoid confusion, we will continue to name the Shc proteins as p46, p52, and p66 according to Pelicci et al.44 (B) Starved TF-1 cells were stimulated for the indicated times with hGM-CSF. Cells were lysed and total cell lysates containing 50 μg of protein were subjected to Western blot analysis using anti-Shc antibodies. The positions of Shc proteins (p46, p52, and p66) are indicated. The arrow above p66 indicates a slower migrating form of p66 detected during hGM-CSF stimulation.