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. 2016 Dec 5;6:38363. doi: 10.1038/srep38363

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Copyright © 2016, The Author(s)

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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(A) Top, western blot on the biotinylated fraction of COS-7 cells co-transfected with the different NEU1-Flag mutants and PPCA (1:2), and probed with a mouse monoclonal anti-Flag antibody. A representative pattern is shown. Bottom, quantification of the relative expression of the NEU1-Flag mutants in the biotinylated fraction by densitometry analysis. Relative expression was calculated as amount of NEU1-Flag mutant recovered in the biotinylated fraction over expression level in cell lysate and normalized to the control (NEU1-Flag). Results are expressed as mean ± SEM of 4 independent experiments. **P < 0.01. (B–D) Confocal images of (B) NEU1-Flag mutants and cathepsin A, or (C) NEU1-Flag mutants and lysosome, or (D) NEU1-Flag mutants and integrin beta 1 distribution in permeabilized COS-7 cells co-transfected with NEU1-Flag mutants and PPCA (1:2). (E) Subcellular distribution of the different NEU1-Flag mutants. Co-transfected COS-7 cells were lysed and subfractioned in heavy membrane (HM), light membrane (LM) and cytosolic (C) fractions. Equal amounts of proteins were subjected to SDS-PAGE and western blotting. Antibody directed against cytochrome c oxidase (COX IV), calnexin (CNX) and actin were used as markers of the different fractions. A representative pattern of three experiments is shown.