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. 2021 Apr 16;12:2286. doi: 10.1038/s41467-021-22642-x

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a, b biotinylation experiments of resting and thrombin-activated platelets WT and Mfsd2bf/f PF4 KO platelets to detect the expression of Mfsd2b in the plasma membrane. Biotin-bound (NeutrAvidin) and unbound fractions were collected from resting and thrombin-activated platelets for detection of Mfsd2b, integrin b3, a plasma membrane marker, and beta-actin, a cytosolic protein. Experiments were repeated at least three times. WT wild-type, KO knockout. c, d Quantification of Mfsd2b protein band from biotin-bound and unbound fractions harvested from resting and activated platelets. The intensity of Mfsd2b bands was normalized to beta-actin bands. Mfsd2b is significantly expressed in the plasma membrane of resting and activated platelets. Data are mean and SD. Each dot represents one mouse (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001. P values were calculated by two-tailed unpaired t-test. e Subcellular fractionation of membrane and cytosolic proteins from resting WT platelets. Proteins from the membrane and cytosolic fractions were probed with Mfsd2b, integrin b3, PF4, and beta-actin antibodies. Similar to integrin b3, expression of Mfsd2b is significantly higher in the membrane fractions. Experiments were repeated three times. f, g Quantification of Mfsd2b and integrin b3 from membrane and cytosolic fractions, respectively. Data are mean and SD. Each dot represents one mouse (n = 8 in f and n = 5 in g). **P < 0.01, ***P < 0.001, two-tailed unpaired t-test is used. h Radioactive S1P in cytosolic and membrane fractions from resting WT platelets. Data are mean and SD. Each dot represents one mouse (n = 5). ***P < 0.001, two-tailed unpaired t-test. i, j mass spectrometry analysis of endogenous S1P in cytosolic and membrane fractions from resting platelets from WT and Mfsd2bf/fPF4 mice. Total S1P levels (sum of all measured S1P species) were used. Data are mean and SD. Each dot represents one mouse (n = 5). **P < 0.01, ***P < 0.001, two-tailed unpaired t-test. The quantification bands (in c, d, f, g) were derived from the same experiments and the blots were processed in parallel. DPM disintegrations per minute.