EPO induces tyrosine phosphorylation and the formation of Grb2/EPO-R complexes. CTLL-EPO-R cells were depleted of cytokine for 8 hours and stimulated with no factor (lanes 1 and 6), 50 U of murine IL-2 per milliliter (lanes 2 and 7), 100 ng of murine IL-4 per milliliter (lanes 3 and 8), 100 ng of simian IL-15 per milliliter (lanes 4 and 9), or 50 U of human EPO per milliliter (lanes 5 and 10) for 10 minutes. After cell lysis, an immunoprecipitation was performed with either a Grb2 polyclonal antibody (lanes 1 through 5) or an anti-amino terminal EPO-R polyclonal antibody (lanes 6 through 10). Immune complexes were resolved by SDS-PAGE and blotted to nitrocellulose. The immunoblot was probed with 4G10 monoclonal anti-phosphotyrosine antibody followed by HRP-sheep anti-mouse IgG. The blot was then stripped and reprobed with a Grb2 polyclonal antibody. Molecular mass standards are indicated. Ab, antibody.