eIF2α regulates CDC25A downstream of JAK2^V617F. (A) CDC25A and eIF-2α protein levels, as well as eIF-2α phosphorylation on ser 51, were analyzed by Western blot in JAK2^WT and JAK2^V617F-expressing FDC-P1–EPOR cells under normal conditions of culture or after 1 hour of treatment with AG490 or JAK2 inhibitor II. (B) CDC25A expression was analyzed by Western blot after 24 hours of treatment with Salubrinal, an inhibitor of eIF2α dephosphorylation, in JAK2^V617F-expressing FDC-P1–EPOR cells or HEL cell lines. β-actin levels were used as control. Western blots are representative of 3 independent experiments. (C) FDC-P1–EPOR JAK2^V617F and JAK2^V617F-positive cells from 3 different patients (PV patient 5, PMF 3, and AML 1) were cultured in the presence or the absence of salubrinal at 75μM. Cells were stained with trypan blue and counted after 48 hours.