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. 2017 Jul 14;7:5399. doi: 10.1038/s41598-017-05637-x

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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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Binding of cocaine results in the enrichment of the R60A mutant relative to co-expressed wt DAT in the filopodia of HEK293 cells. (A) HEK293 cells co-expressing RFP-HA-DAT with YFP-HA-DAT, YFP-HA-DAT-R60A or YFP-HA-DAT-W63A were imaged through 515 nm (YFP, green) and 561 nm (RFP, red) filter channels before and after cocaine treatment (30 min at RT). Maximal z-projections of 5 consecutive x-y-confocal planes are shown. Scale bars, 10μm. (B) Insets represent high magnification images of the regions marked by the white rectangle in (A). Arrows point to examples of peripheral filopodia. (C–E) The Filopodia/FM ratios of YFP and RFP fluorescence intensities in cells co-expressing RFP-HA-DAT with YFP-HA-R60A (C), YFP-HA-W63A (D) or wt YFP-HA-DAT (E) were calculated in experiments exemplified in (A,B) as described in “Methods”. Results are shown as mean ± SEM, n = 10. (F) The YFP/RFP ratio in filopodia was calculated in experiments represented in (A,B). Results are shown as mean ± SEM, n = 10. *p < 0.05, ***p < 0.001. *p < 0.05, ***p < 0.001.