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. 2017 Aug 24;7:9296. doi: 10.1038/s41598-017-09506-5

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© The Author(s) 2017

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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Loss of PS flippase activity in Tmem30a-mutant MEFs. (A) Western blot analysis revealed diminished Tmem30a expression in mutant MEFs. The expression of TMEM30A in mutant MEFs was reduced to 30% of that of control cells. Gel picture was cropped to save space. Full gel picture was listed as Figure S13. P<0.001. (B,C) Annexin V staining showed PS exposure on the mutant MEF cell surface, not on control cells. (D–F) NPD-PS labelling assay revealed loss of PS flippase activity in mutant MEFs. Internalization of NBD phospholipids by control (D) and mutant MEF cells (E). Loss of Tmem30a led to decreased NBD-PS uptake (E). The Y-axis showed numbers of NBD-PS-labelled MEF cells. The X-axis represented NBD-PS fluorescence intensity of intact living cells. Mutant cells exhibited decreased fluorescence intensity, compared to control cells (F). MFI, median fluorescence intensity. N = 3. ***P<0.001.