IFN-α blunts Mpl, JAK2, and STAT-5 phosphorylation in BaF3/mMpl cells and primary murine megakaryocytes.

(A) BaF3/mMpl cells were grown in IL-3 and were washed and starved for 16 hours. Four hours before the addition of 10 ng/mL mTPO, 500 U/mL IFN-α or sham was added to the culture; 10 minutes after TPO was added, the cells were lysed and immunoprecipitated with antibodies to mMpl, JAK2, or STAT5. Immunoprecipitates were size fractionated by denaturing PAGE, and the proteins were transferred and blotted for phosphotyrosine. After probing, the blots were stripped and reprobed for c-Mpl, JAK2, and STAT5. Similar results were obtained from 3 additional experiments. (B) A similar protocol to that in A was followed, except that MK was obtained by elution from an albumin gradient and the cells were starved for 4 hours before the addition of TPO after a 4-hour pretreatment with IFN-α or control. This experiment was repeated twice with similar results. Densitometry of bands in the JAK2 lanes reveal a 20% reduction in JAK2 phosphorylation in MK pretreated with IFN-α and TPO compared with TPO alone.