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. 2019 Aug 29;9:12540. doi: 10.1038/s41598-019-49021-3

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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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Complex formation between Claudin-1, ZO1 and ADAM15 in MDA-MB-231 and T47D cells. Co-localisation of overexpressed ADAM15 (V5, AlexaFluor-488, green) and (a) Claudin-1 (AlexaFluor-594, red) or (c) ZO1 (AlexaFluor-594, red) in MDA/ADAM15 A expressing cells. MDA/FRT and A431 cells do not contain V5 tagged ADAM15 and serve as negative control for V5. A431 were used as positive control for claudin-1cell-cell junction localisation. Co-localisation of ADAM15 (AlexaFluor-488, green) with (c) Claudin-1 (AlexaFluor-594, red) or (d) ZO1 (AlexaFluor-594, red) in T47D cells. Example areas of co-localisation are highlighted with arrows. Scale bar = 10 mm. Immunoprecipitation of (e) ADAM15 (V5) and (f) ZO1 from MDA/ADAM15 A-E expressing cells. V5 IP probed with V5, Grb2 (as a positive control for co-IP with ADAM15) and ZO1. ZO1 IP probed with ZO1 and V5. (g) Representative IF images of Claudin-1 localisation to nuclei in MDA/ADAM15A cells. ADAM15 (ADAM15-ECD, AlexaFluor-594, red) and claudin1 (AlexaFluor-488, green) localization in MDA/ADAM15A, MDA/ADAM15A/sh-non-target and MDA/ADAM15A/shClaudin1 expressing cells. Nuclei visualized by DAPI (blue). Scale bar = 10 µm. (h) Subcellular localization of claudin-1 by fractionation analysis of MDA/ADAM15A expressing cells. HSP90 serves as cytoplasmic, R-cadherin as membrane and Lamin A/C as nuclear control. T = Total cell lysate; C = cytoplasmic fraction; M = Membrane fraction; N = Nuclear fraction.