MARCKS is differentially expressed and phosphorylated during maturation and proplatelet formation in primary murine MKs. (A) MKs were cultured as described and lysed at indicated times. Western blots were quantified relative to the loading control, and then normalized to amount of protein on day 3 (n = 4; ***P < .0001, compared with day 3). (B) Schematic of MARCKS phosphorylation/internalization and dephosphorylation. (C) MKs at day 4 were treated with PMA (PKC activator) at indicated dosages. Percent proplatelet formation was quantified manually and western blots were done to show differential MARCKS phosphorylation status with PMA treatments (n = 4). (D) MKs on day 4 were treated with C2 ceramide (0.1 μM, PP2A activator) or okadaic acid (0.1 μM, PP2A inhibitor). Percent proplatelet formation over time was quantified using the Incucyte imaging system and (E) western blots were done to show differential MARCKS phosphorylation status with treatments (n = 3; *P < .05, **P < .005, ***P < .0001, compared with control).