Figure 1.

Quantification of fluorescent EGF binding to HEK293 cells stably expressing a full-length N-terminal nanoluficerase (NanoLuc) tagged-EGFR measured using NanoBRET. Saturation binding of fluorescently labelled (A) EGF-AF488 and (C) EGF-AF647 in the absence (closed circles) or presence (open circles) of 100 nM unlabelled EGF added simultaneously and incubated for 30 minutes at 37^oC. Saturation experiments were performed in HBSS containing 0.2 % BSA. Displacement of various fixed concentrations of (B) EGF-AF488 or (D) EGF-AF647 by increasing concentrations of unlabelled EGF. Both ligands were added simultaneously, and cells incubated for 60 minutes at 37^oC. The NLuc substrate furimazine (12.5 nM) was added and plates incubated for 5 minutes then luminescence and fluorescence emissions were measured using a BMG Pherastar. Displacement experiments were performed in HBSS containing 0.1 % BSA. Closed bars represent fluorescent EGF alone, with open bars representing vehicle (HBSS/0.1% BSA). Data are combined mean ± SEM from five independent experiments, where each experiment was performed in triplicate.