TIRF Microscopy for Agonist-Driven GRK2 Recruitment

To monitor agonist-driven plasma membrane recruitment of GRK2, we used a TIRF microscopy–based assay described previously (27 (link)). Cells were washed and live imaged in Hepes-buffered saline imaging solution with 135 mM NaCl, 5 mM KCl, 0.4 mM MgCl₂,1.8 mM CaCl₂, 20 mM Hepes, and 5 mM d-glucose adjusted to pH 7.4. Time lapse image series were acquired at 37°C with 488-nm laser excitation using a 100× 1.49 oil CFI Apochromat TIRF objective on a Nikon TIRF microscope operated with NIS Element AR 5.21.03 and equipped with a temperature-controlled chamber (Okolab), perfect focus system, and an Orca Fusion BT sCMOS camera at 5-s intervals. Drugs were added by bath application at concentrations indicated in the figure legends. Protein relocalization was calculated as R(t)/R₀ with R(t) being the fluorescence signal at each time point (t) and R₀ being the mean fluorescence signal of the 10 time points before agonist addition.