Generation of membrane SM in platelets and MKs. (A) Platelets were detected by using phycoerythrin-conjugated anti-CD41 antibody (anti–CD41-phosphatidylethanolamine [PE]) and flow cytometry. (B) Cell surface SM (stained with Venus-lysenin) in CD41⁺platelets from WT (n = 3), SMS1-KO (n = 4), or SMS2-KO (n = 4) mice was analyzed by flow cytometry and quantified as the mean fluorescent intensity (MFI). NS, no staining. (C) SM amounts in platelets from WT (n = 4), SMS1-KO (n = 6), or SMS2-KO (n = 5) mice were measured by using liquid chromatography–tandem mass spectrometry. (D) MKs were extracted from bone marrow cells, then stained with Venus-lysenin and anti–CD41-PE. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI), and images were obtained with confocal microscopy. The fluorescence intensity of Venus-lysenin was quantified by using ImageJ software (National Institutes of Health, Bethesda, MD) and is indicated as arbitrary units (AU). Bars, 20 µm. DIC, Differential interference contrast. Values show the mean ± standard deviation. *P < .05; **P < .005.