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. 2019 Jun 28;10:2864. doi: 10.1038/s41467-019-10891-w

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© The Author(s) 2019

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. 1 — GMAP210 is present in membranes purified from T lymphocytes and containing LAT. a JCAM2.5 LAT-deficient T-cells expressing a chimeric mouse LAT coupled to two Strep-Tag (LAT-TST) were mechanically disrupted. The membrane fraction was then submitted to a floatation gradient on iodixanol. After ultracentrifugation, 10 fractions from top to bottom were collected and submitted to SDS–PAGE and Western blot analysis. b Transmission electron microscopy performed on membranes from fraction 3 showing an immunogold staining for LAT (6 nm gold particles) and GMAP210 (10 nm gold particles). Scale bar: 50 nm. c Fraction 3 prepared from JCAM2.5 (JCAM) or JCAM2.5 expressing LAT-TST (LAT-TST) were prepared as in a. They were mixed with Strep-Tactin Sepharose and submitted to SDS–PAGE and Western blot analysis. The presence of GMAP210, GM130, LAT-TST, and VAMP7 in: the fraction 3 before precipitation; the Strep-Tactin precipitates (StepTactin); and the total lysates obtained in the presence of detergent (Lysate), are shown. Ratios showing the relative expression of the different proteins in JCAM2.5 expressing LAT-TST as compared to the expression in JCAM2.5 are presented under each WB (LAT-TST/JCAM). d Transmission electron microscopy images of fixed Jurkat cells overexpressing LAT showing an immunogold staining for LAT (6 nm gold particles) and GMAP210 (10 nm gold particles); c centriole; g Golgi apparatus. Red arrows show small vesicles presenting both LAT and GMAP210 staining, blue arrows show a bigger vesicle “capping” a centriole. White Scale bar: 1 μm, gray scale bar: 500 nm, black scale bar: 200 nm. Data represent three independent experiments (a) and one experiment (b–d)