IL4R-I242N induced STAT6 but not JAK2 activation in DEV cells. Transduced DEV cells with retrovirus control (empty) or retrovirus expressing IL4R WT or I242N mutant under control of doxycycline (20 ng/mL, 48 hours) were pretreated with PYR or PAC for (A-B) 2 or (C) 24 hours followed by stimulation with (A) human recombinant IL-4 (20 ng/mL) for 20 minutes or (B,C) left UT. (A) Immunoblot analysis of JAKs (JAK1, JAK2, JAK3, and TYK2) phosphorylation was performed following IP with an anti-phosphotyrosine (p-Tyr) antibody. GAPDH analysis in samples before IP was used as a loading control. (B) Phosphorylation status of STAT5 and STAT6 were determined by western blot in DEV cells pretreated with PAC (0.1, 0.25, 0.5, and 1 μM). (C) Flow cytometry analysis of cell-surface expression of CD23 in DEV transduced with IL4R I242N mutant represented as a scatter plot. Numbers represent the mean ± SD of the percentage (%) and GM of CD23 expressing cells and the scatter plots correspond to a representative experiment. IP, immunoprecipitation; PAC, pacritinib; PYR, pyridone 6.