Fig. 6: GRK inhibitor screening using the 7TM phosphorylation assay.

a Control-HEK293, ΔGRK2/3-HEK293, ΔGRK5/6-HEK293 and ΔGRK2/3/5/6-HEK293 cells stably expressing mouse µ-opioid receptor (MOP) were either unstimulated (−) or exposed to 10 µM DAMGO (+) for 30 min at 37 °C. Subsequently, T370, S375, T376 and T379 phosphorylation was determined by western blot analysis. b MOP-expressing Control-HEK293 and ΔGRK2/3-HEK293 cells were exposed to increasing concentrations of DAMGO, and T376 phosphorylation was determined according to the standard protocol. c MOP-expressing Control-HEK293 and ΔGRK2/3-HEK293 cells were exposed to increasing concentrations of DAMGO, and the degree of S375 phosphorylation was determined. d MOP-expressing Control-HEK293 cells were treated with increasing concentrations of the GRK inhibitors LDC9728, LDC8988 or compound101 for 30 min prior to stimulation with 10 µM DAMGO, and T376 phosphorylation was then determined. e MOP-expressing ΔGRK2/3-HEK293 cells were treated with increasing concentrations of the GRK inhibitors LDC9728, LDC8988 or compound101 for 30 min prior to stimulation with 10 µM DAMGO, and S375 phosphorylation was then determined. The data points shown in (b, c) represent optical density read at 405 nm (OD₄₀₅). Data points shown in (b, c) represent optical density readings at 405 nm (OD₄₀₅). Data points shown in (d) were normalized to 10 µM DAMGO stimulation of MOP-expressing Control-HEK293 cells. The data points shown in (e) were normalized to 10 µM DAMGO stimulation of MOP-expressing ΔGRK2/3-HEK293 cells. Western blot images are representatives of n = 5 independent experiments. Immunoassay data are means ± SEM of n = 5 independent experiments performed in quadruplicates. Where error bars are not apparent, SEM was smaller than symbol size.