Cytokine induced Cbl/Crk complexes exceed Cbl/Grb2 complexes in DA-3-EPO-R cells. 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 polyclonal antibody (lanes 1-3), an anti-CrkL polyclonal antibody (lanes 4-6), or an anti-Grb2 polyclonal 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 stripped and reprobed with either Cbl or Grb2 polyclonal antibodies. The exposure periods of the pTyr immunoblot are 3 minutes (lanes 1-6) and 1 minute (lanes 7-9). The migration of EPO-R, Shp2, and Shc were determined by stripping and reprobing the membrane with specific antibodies (data not shown). Molecular mass standards are indicated.