Figure 2.

Steady-state kinetic analysis of JAK2 inhibition by ON044580 (a) ATP does not affect the JAK2-V617F inhibitory activity of ON044580. Recombinant JAK2-V617F (aa 532-1124) was mixed with the indicated concentrations of ON044580 and varying concentrations of ATP up to 5 mM. Kinase assays were performed as described in Figure 1b. The values from individual samples were analyzed and plotted as a function of log inhibitor concentration as described in Figure 1c. (b, c) Effect of substrate concentration on the inhibition of V617F-JAK2 autophosphorylation and GST-Abltide phosphorylation. IC₅₀ curves for V617F-JAK2 kinase activity in the presence of varying concentrations of GST-Abltide substrate and ON044580 were generated as described in (a). The upper band, like that seen in Figure 1b was quantitated for autophosphorylation of JAK2-V617F (b) and the lower band for phosphorylation of GST-Abltide (c).