Figure 6: Barbadin induces the retention of receptor/β-arrestin complexes in clathrin-coated pits (CCPs) at the membrane.

(a,b) Confocal images and colocalization of β-arrestin2, β2-adaptin, and clathrin light chain-(CLC) in CCPs from AVP-stimulated cells expressing V2R. β2-adaptin depleted HEK293 cells (CRISPR-β2Ad-LY5) were transfected with β-arrestin2-mCherry, β2-adaptin-CFP and CLC-YFP, and HA-V2R, and serum-starved for 30 min in the absence or presence of Barbadin (10 μM), before being either left non-stimulated (vehicle) or stimulated with AVP (1 μM) for 2.5 min. Cells were then fixed as described in the Methods section before visualization. Shown in the top panels are black and white micrographs of acquired fluorescent signals from the three-tagged proteins in each channel (red, cyan and yellow), and in colour, are the overlay images. Lower colour insets are close-up images from boxed areas. (b) Colour images represent individual and overlay fluorescent signals taken from different areas, from cells transfected, treated and stimulated as in a. Scale bars in images of whole cells, 10 μm; and insets, 2 μm. Colocalization quantification is presented in Supplementary Fig. 7b–c. (c) Colocalization and quantification of β-arrestin2-mCherry and β2-adaptin-YFP in live HEK293T cells stably expressing V2R. Cells were pretreated with DMSO or Barbadin (10 μM) for 30 min before stimulation with AVP (1 μM) for the indicated durations. Co-localization was quantified using the Pearson correlation coefficient over three independent experiments using 33 or 24 cells for DMSO and Barbadin condition, respectively. Statistical significance of the effect of Barbadin as compared to DMSO was assessed by a two-way ANOVA followed by Bonferroni’s post-hoc tests (*P<0.05).