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. 2017 Aug 31;7:10131. doi: 10.1038/s41598-017-10474-z

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© The Author(s) 2017

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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BRET assay between AT₁R or Ca_V1.2 channel and β-arrestins upon AngII stimulation. Time course of BRET signal ratio from AD-293 cells transfected with β-arrestin₁-RLuc (black circles) or β-arrestin₂-RLuc (grey circles) plus AT₁R-YFP alone (A) or AT₁R-YFP plus the L-type calcium channel (Ca_V1.2, Ca_Vβ and Ca_Vα₂δ) (B). For negative controls (empty circles), AD-293 cells were transfected without the β-arrestins. Time course of BRET signal ratio from AD-293 cells transfected with β-arrestin₁-RLuc (C) or β-arrestin₂-RLuc (D) plus AT₁R and a YFP-tagged L-type calcium channel (Ca_V1.2, Ca_Vβ/YFP and Ca_Vα₂δ). For negative controls (empty circles), AD-293 cells were transfected without the Ca_V1.2 subunit. Red lines correspond to the best fit to a single rectangular hyperbola. The BRET records are averages of at least five independent experiments (n = 5–8). (E) Concentration-response curve of AngII-induced increase in net BRET signal in AD-293 cells transfected with β-arrestin₁-RLuc plus AT₁R and a YFP-tagged L-type calcium channel (Ca_V1.2, Ca_Vβ/YFP and Ca_Vα₂δ), the line correspond to the best fit to Hill equation, n = 3. Mean values ± sem are shown.

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