Tyrosine phosphorylation of signaling proteins in Ba/F3 cells induced by EPO. (A) The indicated cell lines were IL-3 deprived for 6 hours and stimulated with medium alone or EPO (2 U/mL) for 30 minutes. Cell lysates were immunoprecipitated with antibodies as indicated, followed by antiphosphotyrosine immunoblotting with 4G10. (B) Ba/F3 cells expressing EPO R/ABL full length or EPO R/ABL ▵ABD chimeric receptors were IL-3 deprived and stimulated with EPO (2 U/mL) for 0 to 90 minutes. Lysates were immunoprecipitated with antipaxillin (upper two panels) or anti-CRKL (lower panel), followed by immunoblotting as shown. (C) In a similar experiment, lysates were subjected to immunoprecipitation with anti-EPO receptor antibody, followed by sequential immunoblotting with anti-ABL and anti-paxillin.