Ap06779

The localizations and expression of chemerin and CMKLR1 were compared using immunofluorescence (IF) staining. Tissues were stained for 2 h at room temperature in the presence of anti-chemerin (1 : 200, ab72965, Abcam, USA) and anti-CMKLR1 (1 : 200, AP06779, Origene, USA) antibodies, followed by staining for 1 h at room temperature with appropriate Alexa Fluor 488/647-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA). Tissue sections were stained with DAPI for 5 min before imaging.

Total proteins were extracted from the aortic segments using a lysis buffer (Beyotime, China). These lysates were then separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Invitrogen, USA), transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA), and then incubated with primary antibodies against chemerin (1 : 1000, ab103153, Abcam, USA), CMKLR1 (1 : 1000, AP06779, Origene, USA), MMP-2 (1 : 1000, ab86607, Abcam, USA), MMP-9 (1 : 1000, ab38898, Abcam, USA), and GAPDH (1 : 10000, ab8245, Abcam, USA) overnight at 4°C. The next day, the blots were incubated with horseradish peroxidase- (HRP-) conjugated AffiniPure goat anti-rabbit IgG (1 : 5000, SA00001-2, Proteintech, China) or anti-mouse IgG (1 : 5000, SA00001-1, Proteintech, China) for 1h at room temperature. Proteins were visualized using Amersham Imager (Cytiva, USA) and quantified using ImageJ software.