(A and B) Crkl immunoprecipitates from thrombopoietin-treated normal platelets contain a tyrosine-phosphorylated 95- to 100-kD protein that is also recognized by an anti-STAT5 MoAb. Platelets were lysed at the indicated times after thrombopoietin treatment by the addition of an equal amount of a buffer containing 2% Triton X-100 before and after exposure to thrombopoietin (100 ng/mL). Crkl was immunoprecipitated with specific Crkl antisera. Immune complexes were resuspended in SDS-sample buffer. Proteins were separated by 7.5% to 15% SDS-PAGE and transferred onto PVDF membranes. Immunoblots were probed with phosphotyrosine antibodies (A) or a STAT5 MoAb (B) and bands were visualized by chemiluminescence. The asterik in (A) indicates the tyrosine-phosphorylated 95- to 100-kD protein. (C) The same PVDF membrane used in (B) was stripped of the antibody and reprobed with Crkl antisera. Bands were visualized using alkaline phosphatase-conjugated second antibody and NBT/BCIP. (D) Crkl immunoprecipitates do not contain STAT3. Platelets were lysed as in (A) before and after stimulation with thrombopoietin (100 ng/mL for 10 minutes). Crkl or STAT3 was immunoprecipitated with specific antisera as indicated. The membranes were probed with STAT3 antisera (top panel), Crkl antisera (middle panel), or 4G10 (bottom panel). Bands were visualized by ECL. (E) Thombopoietin-induced tyrosine phosphorylation of STAT5 in platelets. Platelets were lysed as in (A) before and after stimulation with thrombopoietin (100 ng/mL for 10 minutes). STAT5 was immunoprecipitated with specific antisera (3 μL of anti-STAT5A and STAT5B each). The membranes were probed with 4G10 (upper panel) or an anti-STAT5 MoAb (lower panel). Bands were visualized by ECL. (F) Association of STAT5 with the Triton X-100–insoluble residue. Platelets were lysed with Triton X-100–EGTA buffer before or after stimulation with thrombin (1 U/mL), with or without stirring. Lysates were separated by high-speed centrifugation into soluble and insoluble residues. Proteins from each fraction were separated by 10% SDS-PAGE and immunoblotted with an anti-STAT5 MoAb (upper panel) or Crkl antiserum (lower pane). (Left lane) Triton X-100–soluble residue of resting cells (1.5 × 10⁷cells); (middle lane) Triton X-100–insoluble residue of resting cells (3.0 × 10⁷cells); (right lane) Triton X-100–insoluble residue from 3.0 × 10⁷ cells, prepared 10 minutes after exposure to thrombin (1 U/mL) with stirring.