Fig. 4
Characterization of receptor-mediated chemokine signaling events. a cAMP assay. HEK293 cells transfected with receptor gene plasmids or an empty vector (V) and pGlo22F plasmid containing a cAMP detector gene were incubated with Glosensor cAMP reagent. Cells were treated with SDF-1α for 10 min prior to isoproterenol (Iso) or no treatment (Veh.). Real-time intracellular cAMP production was measured using luminescence. b Each bar in the graph indicates the percent change between the SDF-1α-pre-treated group versus the maximal value of isoproterenol alone. c Effect of G-proteins on β-arrestin2 recruitment to the receptors. HEK293 cells (Wild) and Gα12/13-deficient HEK293 cells (G12/13 KO) were transiently transfected with the NanoBiT receptor constructs and β-arrestin2. After overnight incubation with and without pertussis toxin (PTx), cells were treated with SDF-1α or not (Veh.) and the luminescence determined. d Graphs of maximum luminescence in each experiment from c. e Receptor-mediated SRE-luc reporter gene expression by chemokines. HEK293 cells were transfected with each receptor constructs (CXCR4, CXCR7, CXCR3) or empty plasmid (V) and SRE-luc plasmids, and ligand-stimulated luminescence was measured (VUF11207 is a CXCR7 agonist, NT = no treatment). Results are the average of three independent experiments. Values are presented as the mean ± SD. *p < 0.05, **p < 0.001