STAT phosphorylation. Lysates were generated from Ba/F3-mMPL cells and purified megakaryocytes before (−) and after (+) TPO stimulation for 15 minutes. Specific STAT proteins were immunoprecipitated from (A) 300 μg of total protein derived from Ba/F3-mMPL cells or (B) 75 μg of megakaryocyte extract. These samples were analyzed by Western blot for changes in phosphotyrosine content (P-Tyr). For megakaryocytes, the exact positions of the immunoprecipitated proteins are indicated by arrows. Equivalent loading in the paired lanes was shown by reprobing with the same antibody used for immunoprecipitation (lower strip).