Immunoblotting

Total cellular protein extraction was performed according to the manufacturer's instructions (Kangcheng Co., Ltd., Shanghai, China). Briefly, the cells were washed with PBS and lysed in Cell and Tissue Protein Extraction buffer on ice for 30 min. Then, the homogenate was collected, centrifuged at 12,000 x g for 30 min at 4°C, and protein amounts were quantified by Coomassie brilliant blue staining. Equal amounts of protein (60 µg) for each sample were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were incubated with either goat polyclonal antibody against GPR17 (1:200; cat. no. sc-74791; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or mouse monoclonal antibody against GAPDH, (1:5,000; cat. no. KC-5G4; Kangcheng Co., Ltd., Shanghai, China) overnight at 4°C. Subsequently, the membranes were treated with IRDye-800CW donkey anti-goat IgG (1:3,000; cat. no. 926-32214; LI-COR Biosciences, Lincoln, NE, USA) or IRDye-800CW goat anti-mouse IgG (1:5,000; cat. no. 610-132-121; Rockland Immunochemicals, Inc., Gilbertsville, PA, USA) for 2 h at room temperature. Images were scanned on an Odyssey fluorescence system (LI‑COR Biosciences). Optical densities of GPR17 (41 kDa) and GAPDH (36 kDa) were quantitatively assessed by Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).