JAK2R564Q is negatively regulated by SOCS3 overexpression and is more sensitive to the JAK2 inhibitor, ruxolitinib, than JAK2V617F. (A) Endogenous levels of SOCS1 and SOCS3 in the JAK2 cell lines, with and without TPO stimulation, shown by western blot. (B) Western blot analysis of protein levels in the JAK2-expressing cells transiently transfected with increasing concentrations of SOCS3 for 24 hours and then starved for a following 4 hours. Numbers represent densitometric quantification of phosphorylated protein levels. (C) JAK2 immunoprecipitation and ubiquitin probe by western blot, after starvation. (D) Cells were grown under starved conditions in the presence of 0 to 1 μM ruxolitinib, a JAK inhibitor. After 48 hours, the number of viable cells was measured as a percentage of the dimethylsulfoxide control. A significant decrease in cell viability, compared with dimethylsulfoxide control, was observed in JAK2R564Q-expressing cells at concentrations of 0.1 nM ruxolitinib and above, whereas cell viability only significantly dropped, compared with the control, in the cells expressing JAK2V617F, with 100 nM and 1 μM ruxolitinib treatment. The IC50 value of JAK2R564Q (15.2 nM) was almost 8 times lower than that of JAK2V617F (114.7 nM).
