EPO fails to activate Shc tyrosine phosphorylation in CTLL-EPO-R cells. Ba/F3 (lanes 1 through 3), Ba/F3-EPO-R (lanes 4 through 6), HCD-57 (lanes 7 and 8), CTLL (lanes 9 through 11), and CTLL-EPO-R (lanes 12 through 14) cells were depleted of cytokine for 4 hours and stimulated with no factor (lanes 1, 4, 7, 9, and 12), 50 U of murine IL-3 (lanes 2 and 5), 50 U of murine IL-2 per milliliter (lanes 10 and 13), or 50 U of human EPO per milliliter (lanes 3, 6, 8, 11, and 14) for 10 minutes. After cell lysis, an immunoprecipitation was performed with an Shc polyclonal antibody. Immune complexes were resolved by SDS-PAGE and blotted to nitrocellulose. The immunoblot was probed with HRP-conjugated antiphosphotyrosine (pTyr) MoAb RC20. The blot was then stripped and reprobed with an Shc polyclonal antibody (Shc immunoblot) or a Grb2 MoAb (Grb2 immunoblot).