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. 2019 Jul 3;7:118. doi: 10.3389/fcell.2019.00118

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Copyright © 2019 D’Souza, Blackburn, Kudlyk, Pokrovskaya and Lupashin.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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EELSs’ cargo is preferentially delivered from the Golgi. 20°C block was utilized to accumulate the RUSH reporter mCherry-Lamp1 in TGN of HEK293T COG4 KO cells. After that, plasma membrane glycoproteins were labeled with GNL-647 and delivery of both endocytic and biosynthetic cargo to the EELSs was analyzed using AiryScan microscopy. (A) Schematic representation of the RUSH assay with a 20°C TGN exit block. (B) Fluorescent images of GNL-647, Cherry-Lamp1 and Lamp2-GFP (EELS marker) immediately after shifting the cells to 37°C and (C) after 3 h of shifting the cells to 37°C. Scale bars are 10 μm.