Fig. 1: Step-by-step flowchart showing the 7TM phosphorylation assay protocol.

a Cells expressing affinity-tagged GPCRs are grown in F-bottom cell culture plates, and upon reaching ≥95% confluency, the cells are exposed to the agonist, antagonist or inhibitor of interest. b The cells are lysed in detergent buffer, and lysates are cleared by centrifugation. c For parallel detection of phosphorylated and total receptors, the lysate of each sample is divided into corresponding wells in U-bottom assay plates. d Anti-tag magnetic beads are added to each well for receptor immunoprecipitation. e Primary phosphosite-specific and phosphorylation-independent antibodies are added to the appropriate wells of each split sample. f A secondary antibody labeled with an enzyme or other detection entity is then added. g An enzyme substrate solution is added for detection, the color reaction is stopped by adding a stop solution, and the optical density (OD) is determined with a microplate reader. (Created with BioRender.com).