CrkL constitutively associates with C3G. DA-3-EPO-R cells were depleted of cytokine for 8 hours and stimulated with no factor (lanes 1, 4, and 7), 50 U/mL of murine IL-3 (lanes 2, 5, and 8) or 50 U/mL of human EPO (lanes 3, 6, and 9) for 15 minutes. Following cell lysis, an immunoprecipitation was performed with either an anti-Cbl antibody (lanes 1-3), an anti-CrkL antibody (lanes 4-6), or an anti-C3G antibody (lanes 7-9). Immune complexes were resolved by SDS-PAGE and blotted to nitrocellulose. The immunoblot was probed with 4G10 monoclonal anti-phosphotyrosine antibody and then reprobed with either a Cbl or C3G polyclonal antibodies. The assignment of EPO-R and Shc was determined by stripping and reprobing the membrane with peptide-specific antibodies (data not shown). Molecular mass standards are indicated.