Akt/PKB and ERK kinase phosphorylation and c-FLIP levels in normal and PV erythroblasts. (A) Western blot analysis of phospho-Akt/PKB levels in normal (N) erythroblasts and in erythroblasts derived from patients with PV harboring the JAK2 V617F mutation (PV). Erythroblasts at day 7 of unilineage erythroid culture were incubated for 48 hours in standard erythroid medium with 3 U/mL EPO or 0.5 U/mL EPO or for 24 hours without EPO. (B) Western blot analysis of phospho-ERK 1/2 levels in normal erythroblasts (N) and in erythroblasts derived from patients with PV harboring the JAK2 V617F mutation (PV), treated as in panel A. (C) Expression of the c-FLIP_long (FLIP_L) and c-FLIP_short (FLIP_S) proteins in normal (N) and PV (PV) erythroblasts. Cells were cultivated for 7 days in standard erythroid medium and treated as in panel A. Results shown in panels A to C are representative of experiments performed with erythroblasts derived from CD34⁺ cells of 5 healthy donors and 5 patients with PV harboring the JAK2 V617F mutation.