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. 2022 Aug 15;13:4728. doi: 10.1038/s41467-022-32390-1

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© The Author(s) 2022

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 Schematic of induced bioluminescence upon GPCR-SmBiT and β-arrestin 2-LgBiT interaction. GPCR, G protein-coupled receptor. HSV-TK, herpes simplex virus thymidine kinase promoter. b Real-time measurement of bioluminescence in HEK cells transfected with non-chimeric β2AR (left) or DREADD-β2AR (right). Baseline measurements followed by ligand application (gray arrow shows onset) of either levalbuterol (left) or CNO (right). Measure of center: Mean of baseline-normalized fold change compared to vehicle (dashed line) in the same experimental repetition. Ribbons: 95% confidence intervals. N = four (Non-chimeric β2AR) or five (DREADD-β2AR) experimental repetitions. Source data are provided as a Source Data file. c Phosphorylation analysis of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in untreated, levalbuterol- or CNO-treated HEK cells transfected with non-chimeric β2AR, DREADD-β2AR, or empty vector. Left: Western blot for pERK1/2 and GAPDH (loading control). The anti-pERK1/2 antibody results in an upper band for pERK1 (44 kDa) and a lower band for pERK2 (42 kDA). Right: Densitometry analysis of combined pERK1/2 normalized to GAPDH. Error bars: standard error of the mean. N = three experimental repetitions. Supplementary Fig. S4 shows full scan Western blot membranes from all repetitions. Source data are provided as a Source Data file.