Impaired Mpl endocytosis in Dnm2-null platelets. (A) Dnm2^fl/fl and Dnm2^fl/flPf4-Cre platelet lysates corresponding to 2 µg of protein were subjected to SDS-PAGE and probed for Mpl and actin as indicated. (B) Dnm2^fl/fl and Dnm2^fl/flPf4-Cre platelets were incubated with 50 ng/mL of TPO for 0 to 20 minutes at 37°C, incubated with a rabbit antibody directed against the extracellular domain of Mpl or control rabbit IgG, incubated with Alexa Fluor 488-labeled goat anti-rabbit IgG antibody, and analyzed by flow cytometry. Data represent anti-Mpl minus control IgG mean fluorescence intensities and are expressed as percentage of resting values (mean ± SEM; n = 4 in each group; **P < .01; ***P < .0001). (C) Plasma TPO levels in Dnm2^fl/fl and Dnm2^fl/flPf4-Cre mice (mean ± SEM; n = 4 in each group; ***P = .0007). (D) Dnm2^fl/fl and Dnm2^fl/flPf4-Cre platelets were incubated with 50 ng/mL of TPO for 0 to 20 minutes at 37°C, lysed, subjected to SDS-PAGE, and probed for phosphorylated JAK2 (pJAK2; Tyr1007/1008) and total JAK2, as indicated. (E) Densitometry analysis of the ratio of pJAK2 to total JAK2 (mean ± SEM; n = 4 in each group; *P < .05; **P < .01; ***P < .0001).