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. 2021 Jul 9;78(17-18):6265–6281. doi: 10.1007/s00018-021-03894-8

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© The Author(s) 2021

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 7 — Various chemerin-derived peptides are internalized by GPR1. a HEK293 cells stably expressing GPR1 internalize Tam-EG(4)-chemerin-9 and the scrambled peptides Tam-EG(4)-scrC9 and Tam-EG(4)-scr2C9, but not Tam-EG(4)-chemerin-7. Cells were stimulated with 1 µM of the respective peptide; data points represent mean ± SEM from at least two independent experiments performed in duplicates. Intracellular accumulation of the labeled peptides over time was measured using a confocal microscopy high content imaging system. b After 20 min, the concentration of Tam-EG(4)-chemerin-9 and both scrambled peptides linearly increases over time. The slope of the linear regression for Tam-EG(4)-chemerin-7 does not significantly differ from zero, and is significantly smaller than the slope for Tam-EG(4)-chemerin-9, Tam-EG(4)-scrC9, and Tam-EG(4)-scr2C9 (p < 0.05, one-way ANOVA with Tukey’s multiple comparisons post-test). Bars represent mean ± SEM. c After stimulation with 1 μM peptide for 30 min, all internalized peptides co-localize with the GPR1-eYFP, demonstrating a receptor-dependent internalization. d Comparing concentration-dependent internalization in stably transfected HEK293-GPR1-eYFP cells and receptor binding to Nluc-GPR1-eYFP demonstrates that peptides need to bind to the receptor to be internalized