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. 2020 Mar 26;3:146. doi: 10.1038/s42003-020-0867-7

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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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Fig. 4 — a Dot-plot of Tango/APJ sdAb cell-displayed library following substrate loading. Cells with high ratio of product/substrate (460 nm/530 nm) were sorted out. The gating window and the percent of gated cell population in the 3rd round cell sorting are shown. b β-arrestin recruitment activity of the parental library and the 1st, 2nd and 3rd round sorted cell populations by Tango/APJ β-arrestin assay. Data are expressed as mean with SD. Statistical analysis was done using unpaired two-tailed t test. The higher product/substrate ratio, the higher agonist activity. c Percentage activation of monoclonal cells by Tango/APJ β-arrestin assay. Stimulation by 100 nM apelin 13 and 0.1% DMSO were set as 100% activation and 0% activation, respectively. d Confocal microscopy of single-cell clones tested for β-arrestin recruitment activity. Negative control: Parental Tango/APJ β-arrestin assay cells. Positive control: Parental Tango/APJ β-arrestin assay cells treated with 15 nM apelin 13. Two representative single-cell clones, 9E10 and 4C6, are shown. A scale bar of 250 µm is shown in each image.