CDC25A is overexpressedand constitutively expressed in JAK2^V617F-positive cell lines. (A) JAK2, STAT5, Akt, and Erk phosphorylation and protein levels were analyzed by Western blotting in FDC-P1–EPOR-JAK2^WT and FDC-P1–EPOR-JAK2^V617F cells. S indicates steady-state culture conditions; −, 12 hours of EPO deprivation; +, 12 hours of EPO restimulation (10 IU/mL). β-actin was used as a loading control. Western blots are representative of 3 independent experiments. Vertical lines have been inserted to indicate a repositioned gel lane. (B) Cell cycle distribution of JAK2^WT and JAK2^V617F-expressing FDC-P1–EPOR cells was analyzed by BrdU and propidium iodide costaining under normal culture conditions. Results are representative of 3 independent experiments. (C) Western blot analysis of CDC25A protein level in JAK2^WT- and JAK2^V617F- expressing BA/F3-EPOR cells. S indicates steady-state culture conditions; −, 12 hours of EPO deprivation; +, 12 hours of EPO restimulation (10 IU/mL). β-actin was used as a loading control. Western blots are representative of 3 independent experiments.