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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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N-glycosylation at the N-terminal region is required for the optimal expression of Gpr176. (A) Gpr176 is N-glycosylated in Flp-In TREx293 cells. Dox-treated Flp-In TREx293-Gpr176 (tet-on) cell extracts were treated with PNGase F or O-glyc and immunoblotted for Gpr176 (upper) and β-Actin (lower). (B) Immunoblots of Dox-treated (+) and untreated (−) Flp-In TREx293 (tet-on) cells expressing WT Gpr176 and the respective mutants for potential N-linked glycosylation sites: N-ter (N4, 11, 17, 26Q), ECL3 (N295Q), and N-ter/ECL3 (N4, 11, 17, 26, 295Q). The same membrane exposed for a longer time is shown on the right. A closed arrowhead indicates the position of the proteins with an N-ter mutation. An open arrowhead points to a minor fraction of Dox-induced WT proteins. Asterisk, nonspecific bands. (C,D) Dox-induced mRNA (C) and protein (D) expression levels of WT Gpr176 and the N-ter, ECL3, and N-ter/ECL3 Gpr176 mutants. Relative Gpr176 mRNA levels were determined by qRT-PCR and normalized to the expression levels of the gene encoding the ribosomal phosphoprotein P0. Values are the means ± s.d. (n = 4). For protein levels, relative band intensities in blots exposed for a longer time (B) were determined using densitometry. ***P < 0.0001 versus WT, one-way ANOVA with Bonferroni post hoc test. Values are the means ± s.d. (n = 3).