Figure 2: Quantification of the ternary complex 2D dissociation kinetics on SSMs by FRET. | Nature Communications

Figure 2: Quantification of the ternary complex 2D dissociation kinetics on SSMs by FRET.

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

Figure 2

(a) Typical assay as monitored by TIRFS-RIf (cartoon depicted at the top): after sequentially tethering (1) IL-4Rα-EC (100 nM) and (2) AF568IL-13Rα1-EC (150 nM, FRET donor) onto a SSM, (3) DY647IL-4 (100 nM, FRET acceptor) was injected to form a ternary complex in an approximate 1:1:1 stoichiometry. The AF568 fluorescence (orange curve) decreases during DY647IL-4 binding due to FRET from AF568IL-13Rα1-EC upon ternary complex formation, while in turn the DY647 fluorescence (red curve) increases (dotted rectangle). After washing out unbound ligand, (4) a large amount of IL-4Rα-EC (1 μM) is tethered onto the membrane prior to (5) fast chasing with unlabelled IL-4 (1 μM) that rapidly occupies all excess IL-4Rα-EC on the membrane. Thus, a 2D exchange of DY647IL-4/IL-4Rα-EC bound to AF568IL-13Rα1-EC by IL-4/IL-4Rα-EC is initiated, which is accompanied by recovery of the donor fluorescence with a rate constant corresponding to . RIf signal is shown in black. (b) The 2D dissociation rate constant was obtained by fitting an exponential function (black line) to the AF568 signal (orange points) within the time window highlighted in a. The RIf signal of IL-4 binding to excess IL-4Rα-EC (black points and dashed line) is shown to indicate the time resolution of this assay. (c) Comparison of the donor fluorescence recovery (that is, dissociation kinetics) obtained for DY647RGA (orange), DY647IL-4 (violet) and DY647KFR (green). Depicted curves show representative single experiments.

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