Fig. 7.
Diffusion of Y1 receptor — β-arrestin sfGFP BiFC complexes measured by FCS. FCS recordings were made from the upper membrane of Y1 A1 (control n = 21, 100 nM NPY treated n = 49 cells), Y1 A2 (NPY n = 64), and Y1 A2ΔLIEFD cells (Y1 A2Δ control n = 35, NPY n = 22), as described in the Materials and methods and illustrated in Fig. 2D. As previously, cells were pretreated at 37 °C for 60 min with vehicle (open bars) or NPY (solid bars), prior to FCS measurements at 22 °C. Pooled data is also compared with Y1sfGFP receptors (Fig. 3), acquired under identical acquisition conditions, for both particle concentrations (A, specific to component τD2) and diffusion co-efficients derived from τD2 (B). Plasma membrane fluorescence in Y1 A2 cells was negligible until these cells were stimulated with NPY, with no FCS possible under control conditions. Significant differences (**P < 0.01, ***P < 0.001; Kruskal–Wallis with Dunn's post test) refer to comparison with Y1 A2 NPY data (A) or Y1sfGFP control data (B).