(A and B) Crkl immunoprecipitates from erythropoietin-stimulated erythroid cells contain a tyrosine phosphorylated 95- to 100-kD protein, which is also recognized by a STAT5 monoclonal antibody. Erythroid cells were lysed by the addition of an equal amount of a buffer containing 2% Triton X-100 before and after exposure to eythropoietin (10 U/mL for 10 minutes). Crkl was immunoprecipitated with specific Crkl antisera. The Crkl immunoprecipitates were divided into two. Tyrosine phosphorylated proteins (A), STAT5 (B, upper panel), and Crkl (B, lower panel) in the Crkl immunoprecipitates were detected as described in Fig 1. (C) Crkl-STAT5 coimmunoprecipitation was inhibited by phenylphosphate (PP, 20 mmol/L) or Crkl immunizing peptide (20 μg/mL). Erythroid cells were divided equally to four samples. Three samples were incubated wtih eythropoietin (10 U/mL for 10 minutes). Crkl was immunoprecipitated with specific Crkl antisera. STAT5 (upper panel) and Crkl (lower panel) in the Crkl immunoprecipitates were detected as described in (A) and (B). (D) Bacterially expressed the SH2 domain of Crkl (GST-Crkl-SH2) binds to STAT5 from erythropoietin-stimulated erythroid cells. Lysates from starved (−) erythroid cells or cells stimulated with 10 U/mL of erythropoietin for 10 minutes (+) were analyzed for binding to GST-Crkl-SH2. Bound proteins were separated by SDS-PAGE, transferred to PVDF membranes and immunoblotted with STAT5 (upper panel) or GST (lower panel) monoclonal antibodies.