Enhanced chemiluminescence western blotting detection kit

Total proteins were extracted using a total protein extraction kit (Vazyme Biotech, Nanjing, China), and concentration was measured using a bicinchoninic acid protein assay kit (Beyotime Biotechnology, Shanghai, China). A total of 20 μg of protein was separated by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. The membrane was incubated in blocking buffer containing 5% nonfat dry milk in Tris-buffered saline and Tween 20 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 0.05% Tween) for 1 h at room temperature. After incubating the membrane with primary antibody (rabbit anti-c-Kit antibody diluted at 1 : 3000) overnight at 4°C and horseradish peroxidase- (HRP-) conjugated goat anti-rabbit antibody (1 : 4000, Abcam) for 2 h at room temperature, protein-antibody complexes were visualized with an Enhanced Chemiluminescence Western Blotting Detection Kit (Beyotime Biotechnology, Shanghai, China) and analysis system (Bio-Rad). β-Actin was detected by the same method as a loading control.

PBMCs and brain tissue lysed on ice for 30 min in RIPA lysis buffer containing phenylmethylsulfonyl fluoride and centrifuged at 12 000 r.p.m. for 15 min at 4°C; the supernatant was immediately transferred to a fresh tube on ice. Protein concentration was measured with the bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China) with a bovine serum albumin standard concentration curve and absorbance readings at 562 nm on a spectrophotometer. Equivalent amounts of protein (30 µg) were separated by 10% acrylamide sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to a polyvinylidenedifluoride membrane that was blocked with 5% nonfat milk and probed with primary antibodies against CB1R, CB2R, NAPE-PLD, FAAH, DAGL-α, MAGL and GAPDH. The membrane was then incubated with horseradish peroxidase-conjugated goat anti-rabbit, and goat anti-mouse secondary antibody (antibodies details seen in electronic supplementary material, table S4). Protein bands were detected with an enhanced chemiluminescence western blotting detection kit (Beyotime Institute of Biotechnology, Shanghai, China). Results were analysed using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA) to obtain the optical density ratio of the target protein to GAPDH. Measurements were obtained for triplicate samples.

The cells were lysed with RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) to extract total protein. Then the total protein solution was quantified by the BCA kit (Beyotime Biotechnology, Shanghai, China). The samples were heated for 5 min at 100 °C before the protein samples were separated by 10% SDS-PAGE gel electrophoresis, the target proteins are transferred from the gel to a PVDF membrane (the PVDF membrane was cut to 2 cm wide to transfer the target proteins instead of using a full-length PVDF membrane to the gels), then the PVDF membranes were washed with Tris-buffered saline containing 0.1% Tween-20 (TBST) and blocked with 5% skimmed milk at room temperature for 2 h. After three times of TBST washing, the membrane was incubated with a special primary antibody at 4 °C overnight. Subsequently, the membranes were washed with TBST and then incubated with the secondary antibody for 1 h at room temperature. The immunoreactive proteins were detected using an enhanced chemiluminescence western blotting detection kit (Beyotime Biotechnology, Shanghai, China) and ChemDoc XRS and quantified with Quantity One software (Bio-Rad, Hercules, CA, USA). All antibodies are from Abbkine (Abbkine Scientific Co., Ltd, Wuhan, China).