Viability curves, immunoprecipitation analysis, and Western blots revealing viability and down-modulation of signaling intermediates downstream of JAK2 with various concentrations of INCB16562 in Ba/F3 isogenic cell lines bearing mutant MPN alleles. (A) Cells bearing mutations which result in constitutive activation of the JAK-STAT signaling pathway (JAK2V617F, JAK2K539L, and MPLW515L) have a lower IC₅₀ compared with Ba/F3 cells bearing BCR-Abl. \E Error bars denote SEM. (B) Immunoprecipitation analysis demonstrated decrease in JAK2 phosphorylation in Ba/F3 isogenic cell lines with INCB16562 treatment. A total of 20 million cells were incubated with either DMSO or INCB16562 for 4 hours, followed by immunoprecipitation as described previously.¹³  (C) Western blots revealing a dose-dependent down-modulation of signaling intermediates in the JAK-STAT pathway after treatment with INCB16562.