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. 2019 Sep 3;9:12706. doi: 10.1038/s41598-019-48971-y

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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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Acute and chronic effect of CSC on CFTR PM expression. (a) The effect of 30 min 200 μg/ml CSC on CFTR PM density in CFBE was measured by cell surface biotinylation with NHS-SS-biotin and immunoblotting of the affinity-purified biotinylated proteins. PM selectivity of biotinylation was demonstrated by complete reversibility of biotin-conjugation with the cell -impermeable reducing agent MESNA (50 mM, left panel). Neither the core-glycosylated CFTR nor cytosolic Hsp90 was susceptible to biotinylation, indicating that covalent labelling of the PM by biotin was selective to the cell surface proteins (right panel). Complex- and core-glycosylated CFTR are indicated by an empty and filled arrowhead, respectively. (b) Quantification of PM resident CFTR-3HA after 30 min of 200 μg/ml CSC treatment with or without 10 min of forskolin stimulation by the biotinylation assay as in panel b or Data are means n = 9–10 (c) CFTR detection by cell surface ELISA with Amplex red substrate. Data are means ± SEM, n = 3–6 (d) Chronic (24 h) CdSO₄ or (e) CSC treatment in CFBE CFTR PM density measured by ELISA with Amplex red assay. Data are means ± SEM, n = 3, *p < 0.05.