EPO and IL-3 activate the formation of Cbl/CrkL complexes. DA-3-EPO-R cells were depleted of cytokine for 8 hours and stimulated with no factor (lanes 6 and 12), 50 U/mL of murine IL-3 (lanes 1-5 and 13) or 50 U/mL of human EPO (lanes 7-11 and 14) for various periods of time as shown. Following cell lysis, an immunoprecipitation was performed with a CrkL polyclonal antibody. Lysate controls from 15 minute stimulations are shown in lanes 12-14. 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 antimouse IgG. The blot was then stripped and reprobed with either Cbl or CrkL polyclonal antibodies. Molecular mass standards are indicated.