Figure 3.

Nb80 preferentially interacts with active β₂AR, whereas Nb1, Nb5, and Nb38 preferentially recognize CCR7 independent of its activation state as assessed by split-luciferase complementation. (a,c) Schematic representation of the split-luciferase complementation assay. Nb-GPCR interactions are determined by reconstitution of Small BiT (SmBiT) and Large BiT (LgBiT) to functional NanoLuc (NLuc) luciferase before and after agonist stimulation and subsequent measurements of luminescence signals generated by the reconstituted luciferase. (b,d) HEK293 cells transiently co-expressing β₂AR (b) or CCR7 (d) fused to SmBiT of NLuc and individual Nb clones fused to LgBiT of NLuc were incubated with coelenterazine H (5µM), the luciferase’s substrate, and after 10 min, stimulated with isoproterenol (iso) (10 µM) (b) or CCL19 (1.5 µg/mL) (d). As control, we transiently co-expressed LgBiT without Nb together with either GPCR-SmBiT. Reconstituted luciferase activity between Nb80 and β₂AR (b) and Nb1 and CCR7 (d), respectively, was set to 100%. Results represent each the mean values of three independent experiments including the standard error of the mean (SEM).