Fig. 5.

Identification of Y1 receptor β–arrestin association using sfGFP BiFC. Live HEK293 cells stably co-expressing Y1 receptor-Gc and either β-arrestin1-Gn (Y1 A1), β-arrestin2-Gn (Y1 A2) or β-arrestin2ΔLIEFD-Gn (Y1 A2Δ) were imaged by confocal microscopy at 37 °C (A). To the right, equivalent images are also shown for cells which instead expressed the mutant Y16A receptor-Gc in combination with β-arrestin-Gn (Y16A A1, Y16A A2). BiFC fluorescence was examined in cells under control conditions, or following 60 min 100 nM NPY treatment. Constant acquisition settings were used for the paired native and 6A mutant cell line images, taken during the same experiment (from n = 2–5). In B, images of the Y1 A1 and Y1 A2 cell lines were acquired using the IX Ultra confocal platereader, following vehicle or 100 nM NPY treatment and fixation. Representative examples are magnified to show 25% of the area of each original BiFC image. In each case the analysis panel shows the identification of nuclei (grey, original H33342 image not shown) and BiFC fluorescent compartments (> 3 μm diameter, white) by the granularity algorithm. This allowed measurement of average granule intensity/cell for each image, from which the quantified data in Fig. 6 was derived.