Receptor Internalization Assay.

Assays for receptor internalization were performed as described in Grimsey et al.⁶⁵ with modifications as described in Zhu et al.⁴² In brief, agonist-mediated receptor internalization was measured using a “live-at-start” method. 3HA-hCB₁ HEK cells were plated (40 000 cells/well) into PDL-coated, clear-plastic 96-well plates (Nunc) and cultured overnight. Culture medium was replaced with DMEM supplemented with 1 mg/mL BSA (assay medium) for 1 h. Primary mouse anti-HA 16B12 monoclonal antibody (BioLegend, San Diego, CA) was diluted in assay medium (1:500), warmed to 37 °C, and then applied to each well for 30 min prior to drug stimulation. Antibody-containing medium was aspirated, and wells were washed, followed by application of prewarmed drug dilutions. At the end of the time course, receptor trafficking was arrested with a 5 min incubation on ice. Well contents were aspirated, and secondary antibody was dispensed (AlexaFluor goat anti-mouse 488, diluted 1:300 in assay medium; Thermo Fisher Scientific, Waltham, MA). Secondary antibody was incubated at room temperature for 30 min, aspirated, and then wells were twice-washed with assay medium. Cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) in phosphate buffer (0.1 M) for 10 min. After fixation, wells were aspirated and washed twice in PBS. Hoechst 33258 (4 mg/mL in Milli-Q water, diluted 1:500 in PBS supplemented with 0.2% Triton-X100/PBS+T) was then dispensed for 15 min. Wells were then washed twice in PBS+T and finally left for storage and imaging in 50 μL/well PBS+T supplemented with 0.4 mg/mL merthiolate (thiomersal).

Microplate imaging was performed using an ImageXpress Micro XLS High-Content System automatic microscope. Images were analyzed using the associated analysis platform MetaXpress v6.2.3.733 (Molecular Devices, San Jose, CA) as previously described.^42,65,66 Inverse MRT was calculated for time-course fluorescence data as, as previously described.⁴²