Fig. 5: Hyper-phosphorylated JAK2 is hyperactive in in vivo.

Ba/F cells expressing JAK2-V617F cells were treated for one hour with ruxolitinib and DMSO, and after one hour of pretreatment, ruxolitinib and DMSO is washed out from the cells with PBS (3 times) and incubated in the absence of inhibitor with indicated time points and lysates were measured for STAT5, AKT and ERK activity (A). Quantification of phsopho-STAT5 was measured with indicated time periods when lysates were prepared from ruxolitinib wash and DMSO wash (B). Similarly, ruxolitinib washed out and DMSO washed out samples were incubated with the indicated time period to measure the c-Myc, PIM1and Bcl-2 levels (C). Ba/F3 cells expressing the JAK2-V617F were treated with 1 μM ruxolitinib or DMSO for a time period of 45 min, and ruxolitinib and DMSO were washed out from cells and incubated for a period of 24-h and measured the cell proliferation using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium (MTS)- based method. Data is shown as mean ± standard deviation (SD) (n = 3). OD—optical density (D). **p < 0.01, *p < 0.05 and n.s., not significant, p > 0.05 by Student’s t test.