Quantitative Western Blotting for Opioid Receptors

Western blotting was performed essentially as previously described (Blackwood et al., 2018 (link)). In brief, soluble protein lysates were prepared in solutions that contained 1x NuPage LDS Sample Buffer (Thermo Fisher Scientific, Waltham, MA, United States), and 1% β-Mercaptoethanol (Sigma, St. Louis, MO, United States). Samples were then boiled and resolved using NuPage 10% Bis-Tris Protein Gels (Thermo Fisher Scientific, Waltham, MA, United States). Proteins were electrophoretically transferred onto Trans-Blot^® Turbo^TM Midi Nitrocellulose membranes using the Trans-Blot^® Turbo^TM system (Bio-Rad, Hercules, CA, United States). Primary rabbit antibodies used were anti-OPRM1 (1:5000, ab17934), anti-OPRK1 (1:10000, ab183825), anti-OPRD1 (1:1000, ab176324), and anti-Cyclophilin B (CYPB) (1:10000, ab16045) purchased from Abcam (Cambridge, MA, United States). Secondary antibodies used were goat anti-rabbit (1:500, Sc-1404) conjugated HRP purchased from Santa Cruz Biotechnology (Dallas, TX, United States). Following secondary antibody incubation, ECL clarity (Bio-Rad, Hercules, CA, United States) was used to detect gel bands on ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, United States), and intensities were quantified with Image Lab version 6.0 (Bio-Rad, Hercules, CA, United States) software.