Tyrosine phosphorylation of Shc in mutant cell lines. Cells were incubated in the absence or presence of 10 ng/mL GM-CSF for 15 minutes. Lysates from 10⁷ cells were immunoprecipitated with anti-SHC antibody and subjected to 6% to 12% gradient SDS-PAGE. Immunoblot analyses were performed with either antiphosphotyrosine, anti-SHC, or anti-GRB2 antibody using the same membrane, as indicated.