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. 2021 Jul 16;12:4357. doi: 10.1038/s41467-021-24574-y

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© The Author(s) 2021

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. 5 — a Confocal microscopy of HEK293T cells expressing HA-US28 and non-US28 targeting intrabody-mVenus (Irr Nb mV), VUN103-mVenus (VUN103 mV) or Nb7-mVenus (Nb7 mV) with (CX3CL1) or without (No CX3CL1) the addition of 30 nM CX3CL1. US28 was stained with an anti-HA Alexa Fluor555 antibody on ice before the addition of CX3CL1 to stain the US28 receptor population on the surface. Cells were fixated 20 min after the addition of CX3CL1 and stained US28 (US28 and different nanobody-mVenus (Nb-mVenus) constructs were visualized. Representative data of two independent experiments. b Saturation BRET with HEK293T cells expressing US28-Renilla luciferase (US28-Rluc) and different ratios of Irr Nb mV, VUN103 mV, or Nb7 mV. Data were plotted as the ratio of nanobody-mVenus constructs (Nb-mVenus) and US28-Rluc (n = 3 independent experiments). c BRET using HEK293T cells expressing US28-Rluc, US28-Rluc, and VUN103 mV or Nb7 mV 30 min after addition of 30 nM CX3CL1 (CX3CL1). BRET signal was normalized to the signal of unstimulated (untreated) HEK293T cells expressing US28-Rluc only at timepoint t = 0 (n = 3 independent experiments). d BRET with HEK293T cells expressing US28-Nluc, Venus-mini-Gα_q, and an inducible FLAG-tagged non-US28 targeting intrabody (Irr Nb-FLAG), FLAG-tagged VUN103 (VUN103-FLAG) or FLAG-tagged Nb7 (Nb7-FLAG) 30 min after addition of 100 nM CX3CL1 (CX3CL1). Expression of FLAG-tagged intrabodies was induced using 100 nM of tebufenozide, one day prior to read-out. BRET signal was normalized to the signal of the unstimulated (Untreated) HEK293T cells (n = 3 independent experiments). All data are plotted as mean ± SD. Statistical analyses were performed using an unpaired two-tailed t-test. ns, p > 0.05. Source data are provided as a Source Data file.