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. 2020 Mar 10;10:4429. doi: 10.1038/s41598-020-61370-y

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© The Author(s) 2020

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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PNGase F-treated Gpr176 retains its cAMP-repressive activity. (A) Schematic experimental design for the glycosidase-treated cell suspension GloSensor assay. Dox-treated (+) and untreated (−) cells were resuspended in HBSS with or without PNGase F for 1 h before the GloSensor cAMP assay. (B) Fsk-induced GloSensor activity traces in PNGase F-treated and untreated Flp-In TREx293-Gpr176 (tet-on)/GloSensor (constitutive) cell suspensions. The arrow indicates the start of Fsk treatment. RLU, relative light units. Values (means ± s.d., n = 9 for each data point) are plotted relative to the average peak value obtained in Dox-untreated cells. Insets indicate relative AUC values (means ± s.d., n = 9; ***P < 0.0005, two-tailed unpaired t-test). (C) Immunoblots of representative PNGase F-treated and untreated Flp-In TREx293-Gpr176 (tet-on)/GloSensor (constitutive) cells. The cell suspension samples were immunoblotted for Gpr176 (upper) and Hsp70 (lower). Hsp70 served as a loading control.