Tyrosine phosphorylation of c-Mpl, JAK2, and STAT5 in UT-7/TPO cells stimulated by JTZ-132. UT-7/TPO cells (2.5 × 106 cells/tube) stimulated with JTZ-132 or rhTPO for 10 minutes were lysed by the addition of lysis buffer. (A) Tyrosine phosphorylated proteins were immunoprecipitated with py99, separated by SDS-PAGE, and transferred onto nitrocellulose membrane. The immunoblots were probed with specific antibody of c-Mpl and detected by a horseradish peroxidase–coupled second antibody. (B) JAK2 was immunoprecipitated with specific anti-JAK2 antisera. Immune complexes were separated by SDS-PAGE and transferred onto nitrocellulose membrane. The immunoblots were probed with py99 and horseradish peroxidase–coupled second antibody. (C) Cell lysates were separated by SDS-PAGE and were immunoblotted with antiphospho-STAT5A/B antibody and probed with horseradish peroxidase–coupled second antibody. In all assays, 20 μL of each sample was applied to SDS-PAGE separation. The proteins were visualized by using chemiluminescence detection system.