Expression of JAK2R564Q causes increased intracellular signaling in cell lines and patients. (A) JAK2 activity determined by in vitro kinase assay, based on the ability of the kinase to phosphorylate an IκB-α substrate. (B) After starvation overnight and treatment with/without 10 ng/mL TPO for 5 minutes, cells were lysed and proteins subject to MPL pull-down and western blotting with p-Tyr probe to show levels of phosphorylated MPL in the cell lines. In the absence of TPO, MPL is not phosphorylated by WTJAK2 but is phosphorylated by each of the 3 JAK2 mutants. (C) After starvation overnight, western blot analysis shows increased levels of phosphorylated JAK2 and STAT1, STAT3, and STAT5 in the mutant cell lines, in the absence of, and at low concentrations of TPO treatment of 5 minutes. (D) Western blot analysis of the phosphorylation status of signaling proteins downstream of JAK2 in starved conditions. (E) Platelets were isolated from 3 members of the family with JAK2R564Q mutation and subject to western blot analysis.
