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. 2017 Jun 26;7:4258. doi: 10.1038/s41598-017-04053-5

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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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Side view of Dlgh1 and PKC-θ intracellular distribution in tTreg, iTreg and Teffs. The cells were activated using lipid bilayers as mentioned above, for 10 min at 37 °C, fixed and processed for Dlgh1 and PKC-θ immunofluorescence. Cells were imaged using line scanning confocal microscope, and images were 3D projected to reveal intracellular distribution of aforementioned proteins. Anti-CD3 (pseudocolored red) marks synaptic and proximal areas, while the proteins have been pseudocolored green. Note the enriched distribution of Dlgh1 at the proximal and of PKC-θ towards distal side in tTreg (left panels), and a reduction of this distribution in iTreg (middle panels). The panels show representative images. Dlgh1 and PKC-θ staining was quantified by calculation of average fluorescence intensity in cells. Data are representative of two different experiments. P values were calculated using Mann-Whitney test.