Megakaryocytes and platelets express Mnk1 protein. (A) Washed platelets (Plts), megakaryocytes (Megs), and white blood cells (as a control) from both healthy human donors and WT C57Bl/6 mice were lysed and analyzed for total Mnk1 and Mnk2 protein expression by western blotting. β-Actin was used as a loading control. Images representative of n ≥ 3 independent experiments. (B) CD34⁺ megakaryocytes derived and cultured from human cord blood were lysed after stimulation with or without TPO (100 ng/mL) on culture day 13 for 15 minutes. Lysates were analyzed for phosphorylated Mnk1 (p-Mnk1, left) and phosphorylated eIF4E (p-eIF4E, right) by western blotting. β-Actin was used as a loading control. Images representative of n ≥ 3 independent experiments. (C) Washed human platelets were left alone (0 minutes) or stimulated with AYPGKF (100 μM), collagen (5 μg/mL), or 2MeSADP (50 nM) for 5 and 10 minutes. Samples were analyzed for p-Mnk1 (left) and p-EIF4E (right) by western blotting. β-Actin was used as a loading control. Images representative of n ≥ 3 independent experiments. (D-E) Washed human platelets were left unstimulated (US) in DMSO vehicle control or treated with different concentrations of the Mnk1 inhibitor CGP 57380 for 5 minutes. Then, platelets were stimulated with AYPGKF (100 μM, 5 minutes). Cells were then lysed and analyzed for p-eIF4E and p-4EBP1 by western blotting. β-Actin was used as a loading control. Quantitative analysis of p-eIF4E and p-4EBP1 normalized to β-actin are shown in the bar graphs to the right. Western blot images are representative of n ≥ 3 independent experiments. (F-G) Washed human platelets were left US or treated with CGP 57380 (10 μM) for 5 minutes. The cells were then left alone in DMSO (Veh) or stimulated with AYPGKF (100 μM), collagen (5 μg/mL), or 2MeSADP (50 nM) for 5 minutes and probed for p-eIF4E by western blotting. β-Actin was used as a loading control. Quantitative analysis of p-eIF4E normalized to β-actin is shown in the bar graph to the right (∗P < .05). Western blot images are representative of n ≥ 3 independent experiments. (H) The expression of total Mnk1 protein in platelets from either WT or Mnk1 KO mice was analyzed by western blotting. β-Actin was used as a loading control. Western blot images are representative of n ≥ 3 independent experiments. (I) Cultured bone marrow megakaryocytes from either WT or Mnk1 KO mice were left alone or stimulated with TPO (100 μM) for 15 minutes and then analyzed for p-Mnk1 (top) and p-eIF4E (bottom) by western blotting. β-Actin was used as a loading control. Western blot images are representative of n ≥ 3 independent experiments. (J) Washed platelets from either WT or Mnk1 KO mice were left unstimulated or stimulated with AYPGKF (100 μM) or collagen (5 μg/mL) for 5 and 10 minutes. Platelets were analyzed for p-Mnk1 (top) and p-eIF4E (bottom) by western blotting. β-Actin was used as a loading control. Images are representative of n ≥ 3 independent experiments. (K) Quantitative analyses of p-Mnk1 (top) and p-EIF4E (bottom) normalized to β-actin (∗P < .05). M, marker; NS, not significant.