Figure 4: Cellular binding and activity of different IL-4 agonists and IL-13. | Nature Communications

Figure 4: Cellular binding and activity of different IL-4 agonists and IL-13.

From: Ligand-induced type II interleukin-4 receptor dimers are sustained by rapid re-association within plasma membrane microcompartments

Figure 4

(a) Live-cell single molecule localization and tracking of DY647KFR bound to endogenous cell surface IL-4Rα and IL-13Rα1 of a HeLa cell. The underlying cell is indicated by the grey background; single molecule trajectories are depicted as red lines (see also Supplementary Movie 2). Scale bar: 10 μm. (b) Cell surface density of bound IL-4 variants and IL-13 quantified by single molecule localization. As negative control, binding of DY647IL-4 after preincubation with 20 nM unlabelled KFR was quantified. (n≥10 cells for IL-13, IL-4, RGA and KFR, n=5 cells for DN and ctrl.) (c) Diffusion properties of cell-bound DY647DN (blue) and DY647KFR (green) presented as step-length distributions, which were fitted (dashed lines) by considering two components corresponding to a slow and a fast mobile fraction. Shift to lower mobility by KFR indicates ternary complex formation in contrast to DN, which does not interact with IL-13Rα1. (d) Comparison of the step-length histograms for DY647IL-13, DY647IL-4, DY647RGA, DY647KFR and DY647DN. Fitted curves in c,d are based on data from n>350 single-molecule trajectories (n=200 for DN) with minimum length of 150 frames. Individual step lengths were determined for a time lapse of 32 ms (1 frame) and histogramed as depicted in Supplementary Fig. 14B. (e,f) STAT6 phosphorylation activity of different agonists in HeLa cells analysed by phospho-flow cytometry. (e) Dose–response curves of STAT6 phosphorylation observed after stimulation for 15 min. (f) Kinetics of STAT6 phosphorylation after stimulation with 1 μM of agonist. Data points in e,f represent mean±s.d. of three independent experiments.

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