In vitro phosphorylation of VLPQDKEYYKVKEPGE substrate. (A) Dose-dependent effect of GM-CSF–induced activation of JAK2 in ECs (•) and U937 cells (⋄). Cells were stimulated for 5 minutes with increasing amount of cytokine. Anti-JAK2 immune-complexes (•, ⋄) or immune complexes obtained with nonimmune rabbit serum (○), in triplicate, were incubated in kinase buffer containing 5 μg of peptide and processed as described in Materials and Methods. (B) Time course of GM-CSF (50 ng/mL)–dependent activation of JAK2 in ECs. (C) Effect of stimulation of JAK2 catalytic activity in ECs treated for 5 minutes with GM-CSF (50 ng/mL), heat-inactivated GM-CSF (50 ng/mL), GM-CSF (50 ng/mL) neutralized by specific antibody or by nonimmune serum, and IL-5 (50 ng/mL). Mean ± SD of three samples in one experiment representative of three performed.