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. 2021 Jul 1;11:13673. doi: 10.1038/s41598-021-93117-8

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© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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AQP6 expressed in transient heterologous transfection constructs rarely reaches the plasma membrane. (A) The N-terminal tGFP-labeled AQP6 construct is shown (tGFP-AQP6). The peptide linker is shown in blue and the C-terminal 10× Histag in black. (B) The C-terminal GFP-labeled AQP6 construct is shown (AQP6-tGFP). The peptide linker is shown in blue and the C-terminal 10× Histag in black. (C) Transient transfection of an AQP6-tGFP construct in HEK293 cells yields very poor to no expression. (D) Transient transfection of tGFP-AQP6 construct in HEK293 cells improves expression but most of the expressed protein is retained in the ER. Scale bars (C, D): 5 µm. (E) Very rarely, the tGFP-AQP6 construct localizes to the PM (white arrows) in HEK293 cells. Scale bar: 3 µm. Cells were observed microscopically using an upright Olympus BX51WI equipped with 100 × 1 NA objective, and images were captured using a Grasshopper3 CMOS camera (FLIR, Richmond, BC, Canada) controlled by Leica LAS X version 3.5 software (available at: https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/). All figures were created using Adobe Illustrator CS6 (available at: https://adobe.com/products/illustrator), under an Adobe Inc., Creative Cloud Desktop 2019 shared device license to Case Western Reserve University (CWRU) that operates until 3/31/2022.