2.22. GRK2 kinase assay

GRK2 kinase enzyme was detected by ADP-Glo Kinase Assay Kit and performed the experiment as per manufacturer's protocol. The GRK2 inhibition assay was performed with the ADP-Glo system using 200 ng of GRK2, 0.5 μg of GRKtide substrate, 25 μmol/L ATP, and the different concentration of CP-25 or GSK180736A for 120 min. As previously described38, after the initial reaction, ADP-Glo reagent was added to the reaction and allowed to incubate for an additional 40 min. Lastly, the kinase detection reagent was added and allowed to incubate for 30 min, and the luminescence was measured with automatic microplate reader (TECAN GENIOS). All data was analyzed, and inhibition curves were fit via GraphPad Prism software.