Figure 4.

Effect of EGFR ligands on the binding of fluorescent (A) Q44c-HL488 (14.6 nM) and (B) Q86c-HL488 (12.5 nM) to full-length N-terminal nanoluciferase-EGFR stably expressed in HEK293 cells. Cells were treated with nanobodies and EGFR ligands simultaneously and incubated for 30 minutes at 37^oC. Experiments were performed in HBSS containing 0.2 % BSA. 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. Blue bars represent BRET ratios obtained for total Q44c-HL488 or Q86c-HL488 in the absence of competing ligand, whereas red bars represent those measured for HBSS/0.2% BSA buffer alone (basal). Data are combined mean ± SEM from five independent experiments, where each experiment was performed in triplicate.