2.4 Western blot
We used a protein extraction kit (Applygen Technologies, Inc.) to
extract protein, followed by quantification using the Bio-Rad protein
assay kit (Bio-Rad Laboratories, Inc.). Next, the protein samples (20 μg
protein/lane) were separated by 12.5% SDS-PAGE and transferred to a
nitrocellulose membrane (Bio-Rad Laboratories, Inc.). The membranes were
blocked with 2.5% nonfat milk at 37℃ for 1 h, followed by overnight
incubation with anti-CysLTR1 (rabbit; 1:1000; PA2310; Innovagen),
anti-CysLTR2 (rabbit; 1: 2000; PA2320; Innovagen), and anti-CGR-α
(mouse; 1:500; ab3580; Abcam) antibodies at 4℃. Next, the membranes were
exposed to horseradish peroxidase-conjugated and anti-human IgG
secondary antibodies (1:200; Sigma-Aldrich; Merck KGaA) for 1 h at 37℃.
The immunoreactive bands were detected using an enhanced
chemiluminescence western blotting system (Thermo Fisher Scientific,
Inc.).