Anti cb1

To assess the levels of CB1 or CB2 receptor protein expression, whole pancreatic islets were homogenized in buffer containing protease inhibitors and centrifuged at 600 × g at 4°C for 10 min. The supernatant was centrifuged at 39,000 × g at 4°C for 15 min. The resulting supernatant corresponded to the cytosolic protein fraction and the precipitate to the membrane protein fraction. The resuspended fractions of tissue homogenates (30 μg protein) were mixed 1:1 with Laemmli buffer and heated (95°C, 5 min) before loading onto a 0.75-mm thick gel. The samples were electrophoresed (150 V, 2 h), and protein was transferred onto a PVDF membrane (Immobilon E, Millipore) at 150 mA for 1 h at 4°C. The membrane was stained with Ponceau’s S Red and photographed (G12 camera, Canon),^{50 (link)} and each lane was then cut for subsequent incubation. The membrane was washed and incubated with PBS 1X – Tween20 0.3%, 10% nonfat dry milk, and 2% goat normal serum for 30 min at 20°C, and then incubated with anti-CB1 (1:1500; Cayman Chemical Company, cat 10006590), or anti-CB2 (1:500; Santa Cruz Biotechnology, Inc., sc-25494) overnight at 4°C. The blot was washed with PBS-Tween20, incubated for 1 h at 20°C with goat anti-rabbit IgG horseradish peroxidase conjugate (1:2000), and developed with diaminobenzidine (0.5 mg/mL in PBS plus 0.3 μL/mL 30% H₂O₂).