Ab103153

Peroxidase/anti‐peroxidase staining was conducted, as previously described,21 on frozen sections of human histologically normal blood vessels, LV, lung, and kidney (all n=3) using mouse anti‐chemerin (1:300; ab72965; Abcam, Cambridge, MA); rabbit anti‐CMKLR1 (1:500; ab64881; Abcam), or rabbit anti‐GPR1 (1:300; ab150536; Abcam). To identify cellular distribution, dual‐labeling immunofluorescent staining was executed, as previously described,21 with frozen sections of human histologically normal blood vessels (n=3) or human ASMCs (n=3). Rabbit primary antiserum against human CMKLR1 (1:100), GPR1 (1:50), or chemerin (1:25; ab103153; Abcam) was applied together with either a mouse antihuman von‐Willebrand factor (vWF) antibody (1:50; Dako, Carpinteria, CA), or mouse antihuman smooth muscle alpha actin (SmαA; 1:100; Dako). Secondary antibodies, Alexa Fluor 633 goat antirabbit immunoglobulin G (IgG; 1:200) and Alexa Fluor 568 goat antimouse IgG (1:200; both from Life Technologies, Carlsbad, CA) were used to avoid the autofluorescence of human vessels in the 488‐nm channel.
All images were processed in Fiji,22, 23 for background subtraction, using the rolling ball method, histogram stretching, and merging of channels.

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.