Radioligand Binding Assay Protocol

Radioligand binding was generally carried out as reported in our previous work (e.g., [18 (link),31 (link),32 (link),33 (link)]). Briefly, 30 μg of cell membrane protein was combined with 0.89 nM of ³H-diprenorphine (PerkinElmer, #NET1121250UC) and concentration curves of experimental ligand or reference drug (see Figure Legends for details) in a 200 μL reaction volume. The reactions were incubated at room temperature for 1 h; then, they were collected onto GF/B filter plates (PerkinElmer) using a Brandel Cell Harvester. Then, the plates were read on a PerkinElmer Microbeta2 scintillation counter. The resulting data were normalized to vehicle-alone treatment (100%) and non-specific binding with 10 μM reference compound (0%) and fit to a one-site competition binding model using GraphPad Prism 9.0. The previously measured K_D of the diprenorphine in each cell line [34 (link)] was used to calculate the K_I of each ligand at each receptor.