CHO cell membrane preparation and radioligand displacement assay

Cells were thawed, diluted in Tris buffer (50 mM Tris–HCl and 50 mM Tris–base) and homogenized in a 1 mL hand-held homogenizer ^19,51. Untransfected, hCB1R, and hCB2R CHO-K1 cell membranes were collected by cavitation in a pressure cell, and sedimented by ultracentrifugation, as originally described in Bolognini et al. ⁴¹. Pellets were resuspended in TME buffer (50 mM Tris–HCl, 5 mM MgCl₂, 1 mM EDTA, pH 7.4) and protein concentration was measured via the Bradford method (Bio-Rad Laboratories, Mississauga, ON).

Assays have been described in detail previously and are summarized here ^19,52. Saturation binding experiments were conducted with 0.1–100 nM [³H]CP55,940 and Tris binding buffer (50 mM Tris–HCl, 50 mM Tris–base, 0.1% BSA, pH 7.4, 2 mL) ^19,52. Saturation binding experiments utilized CHO-K1 cells expressing either hCB1R or hCB2R (total bound) and untransfected CHO-K1 cells (non-specific bound). Specific binding curves were calculated as the difference between total and non-specific for each concentration of [³H]CP55,940 used and fit to a one site total binding non-linear regression (GraphPad, Prism, v. 9.0) (Supplementary Fig. ¹). Competition binding experiments were conducted with 0.7 nM [³H]CP55,940, SCRAs as described, and Tris binding buffer (50 mM Tris–HCl, 50 mM Tris–base, 0.1% BSA, pH 7.4, 2 mL) ^19,52. Radioligand binding began with the addition of CHO-K1 cell membranes (50 µg protein per sample). Assays were performed for 120 min at 37 °C and stopped by the addition of ice-cold Tris binding buffer, followed by vacuum filtration using a 24-well sampling manifold (Brandel Cell Harvester; Brandel Inc, Gaithersburg, MD, USA). Brandel GF/B filter paper was soaked with wash buffer at 4 °C for at least 24 h. Each filter paper was washed six times with a 1.2 mL aliquots of Tris-binding buffer, then air-dried overnight and submerged in 5 mL of scintillation fluid (Ultima Gold XR, PerkinElmer). Liquid scintillation spectrometry was used to quantify radioactivity. For competition binding experiments, specific binding was equal to the difference in radioactivity with or without 1 µM unlabelled CP55,940.