Fig. 5. Intrabody30 sensor efficiently recognizes βarr1 conformation induced by V₂R^T360A.

a Schematic representation of NanoBiT-based assay to measure Ib30 reactivity to βarr1. b Recognition of βarr1 by Ib30 upon stimulation of V₂R^WT or V₂R^T360A. HEK-293 cells expressing the indicated receptor, βarr1 and Ib30 constructs were stimulated with varying doses of AVP for 30 min, followed by the measurement of luminescence (mean ± SEM; n = 4 independent experiments; normalized with luminescence signal for V₂R^WTat maximal ligand dose as 100%; Two-way ANOVA, Tukey’s multiple comparisons test; ns = non-significant). c βarr1 and Ib30 are colocalized upon agonist-stimulation of V₂R^T360A. HEK-293 cells expressing V₂R^T360A together with βarr1-mCherry and Ib30-YFP were stimulated with AVP (100 nM) for indicated time-points followed by visualization using confocal microscopy. Micrographs are representative of three independent experiments (Scale bar 10 μm). Uncropped micrographs are available in the Source Data file. d Line-scan analysis of the indicated regions from confocal micrographs confirms the colocalization of βarr1 and Ib30. e Expression of Ib30 drives endosomal localization of βarr1 for V₂R^T360A. HEK-293 cells expressing V₂R^WT or V₂R^T360A together with βarr1-YFP were stimulated with AVP (100 nM), and the localization of βarr1 was monitored using confocal microscopy (Scale bar 10 μm). f The effect of Ib30 on localization of βarr1 as assessed by manually scoring HEK-293 cells from multiple fields in three independent experiments. Captured confocal images were grouped in two classes i.e., 1–8 min and 9–30 min post-agonist stimulation to monitor membrane and endosomal localization, respectively. The bar graphs indicate the % of cells showing βarr localization at the surface or in endosomal punctate structures in more than 500 cells for each condition collected from different field of views of three independent transfections and imaging experiments. Source data are provided as a Source Data file.