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. 2019 Oct 23;10:4836. doi: 10.1038/s41467-019-12755-9

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© The Author(s) 2019

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. 3 — Weak, transient interactions with Gα peptides increase β₂-AR priming. a Schematic list of reagents used to measure Gα peptide–GPCR interaction. b Representation of affinity pull-down and release assay for monitoring the interaction strength of Qp with β₂-AR-mCer. c mCer fluorescence quantification of supernatants estimate extent of bioQp and bioSp binding to the receptor. Only 13% of β₂-AR-mCer was specifically pelleted by binding to bioQp and all the bound receptors were completely released by excess Qp in 30 s. bioSp bound to 45% of the receptor and most of it was released by an excess of the Sp. Values are mean ± SD from three independent experiments with three repeats per experiment (n = 3). d Left, ΔFRET assay for agonist-stimulated β₂-AR activation, in the presence of Gα peptides, using native β₂-AR-Sp sensor membranes. Right, measuring the effect of Gα peptide (Sp/Qp) concentration on ISO-stimulated (100 μM) ΔFRET ratio of β₂-AR-Sp sensors. Critical concentration (C_C) to elicit response is indicated. Values are mean ± SD from four independent experiments with at least four repeats per experiment (n ≥ 4). e Quantification of the extent of photocleavage of PC-Qp by MALDI-MS. UV irradiation of full-length PC-Qp in the presence of native membranes causes the concentration of the full-length PC-Qp to decrease to 4.58 μM, which is less than the critical concentration (C_C) required for GPCR priming. Values are mean ± SD from three experiments. f Representation of a competition assay to monitor displacement of PC-Qp (fragments) from native membranes expressing β₂-AR-mCer. g Presence of Qp or intact PC-Qp competes with bioQp for binding to the receptor. Thirty seconds after UV irradiation, PC-Qp no longer competes with bioQp for interaction with the receptor. Values are from at least 12 replicates across 3 experiments Box-and-whisker plots: center line is median, box ends are upper and lower quartiles, whisker ends are 1.5 × interquartile range (IQR) from four independent experiments with at least three replicates per experiment (n ≥ 4). Statistically significant differences were assessed by a one-way ANOVA, followed by Tukey’s post-hoc test. Significance is denoted by asterisks, NS-not significant, ***p < 0.001, **p < 0.01