JAK phosphorylation. Specific JAK proteins were immunoprecipitated and analyzed by Western blot for changes in phosphotyrosine content before (−) and after (+) TPO stimulation for 15 minutes. (A) In the three cell lines tested, 300 μg of total protein was used for each immunoprecipitation. The blots were stripped and reprobed to show equal precipitation and loading of JAK2 and TYK2 (see strip below). (B) For purified megakaryocytes, 75 μg of total protein was used for each immunoprecipitation. Tyrosine phosphorylated JAK2 and TYK2 are indicated by arrows. A slightly smaller band, most prominent in the JAK1 lane, was nonspecifically precipitated and was found not to be a JAK protein when the blots were reprobed with JAK-specific IgG. In two similar experiments, this artifactual band was not seen.