Anti aggf1

Carotid arteries and ascending aortas were excised, fixed with 4% paraformaldehyde overnight, paraffin-embedded, and sectioned (3 mm). Cross-sections of arteries were stained by haematoxylin and eosin (H&E), Sirius red or antibodies (immunostaining) as described^{22 (link),25 (link),27 (link),47 (link),51 (link),52 (link)}. For immunostaining, the sections were deparaffinized, and antigen was retrieved. 3% hydrogen peroxide was used to block peroxidase. After washing with PBS buffer for three times, the sections were blocked with 3% BSA for 30 minutes. The primary antibody was added and incubated with sections overnight at 4 °C on a shaker, followed by addition and incubation with the secondary antibody at room temperature for 60 minutes. After washing with PBS buffer for three times, the DAB chromogenic solution (Cat number G1212, Servicebio, China) was added, and the nuclei were counterstained with hematoxylin. The slides were dehydrated, mounted, and photographed under a florescence microscope or a confocal microscope. The primary antibodies used in this study include anti-AGGF1 (Cat number 11889-1-AP, Proteintech, America), anti-α-SMA (Cat number GB111364, Servicebio, China), anti-MCP1 (Cat number GB11199, Servicebio, China), and anti-CD68 (Cat number GB11067, Servicebio, China).

Western blot analysis was carried out as described previously (28 ). Protein extracts from cultured MOVAS-1 cells or mouse carotid artery samples were prepared with Western-IP lysis buffer (Beyotime) supplemented with proteinase inhibitor cocktail (Roche). The primary antibodies used for Western blotting include: anti-AGGF1 (Proteintech, 1189-1, 0.7 μg/ml), anti-MYH11 (Proteintech, 21404-1, 0.7 μg/ml), anti-α-SMA (Proteintech, 55135-1, 0.6 μg/ml), anti-SM22 (Proteintech, 10493-1, 0.33 μg/ml), anti-GAPDH (Proteintech, 60004-1, 0.166 μg/ml), anti-pMEK1/2 (Bioss, bsm-52176R, 1 μg/ml), anti-pELK (Bioss, bs-10154R, 1 μg/ml), anti-ERK1/2 (Abcam, 184699, 1.2 μg/ml), anti-pERK1/2 (Abcam, 223500, 1:1000 dilution), anti-integrin α5 (Proteintech, 10569-1, 0.5 μg/ml), anti-integrin α7 (Abclonal, A14246, 1:500), and anti-integrin α8 (Abclonal, A13056, 1:500). The secondary antibodies include a goat anti-rabbit IgG or a goat anti-mouse IgG (HRP-conjugated, 1:20,000) from Biofly. Images from Western blot analysis were captured using a ChemiDoc XRS (Bio-Rad Laboratories) with the SuperSignal West Pico Chemiluminescent Substrate (Pierce Chemical Co) and further analyzed with Gel-Pro analyzer.