Ab4441

The upper parts of the hearts containing the aorta root were harvested, fixed in 4% paraformaldehyde and paraffin-embedded. Sections of the aorta sinus in the aorta root (5-μm thick) were created and used for H&E staining or immunohistochemical staining. For H&E staining, the plaque areas of sections were stained by H&E staining and calculated in a blinded manner by using IPP6 software. For immunohistochemical staining, the sections were deparaffinized, and quenched with 3% (vol./vol.) hydrogen peroxide to remove endogenous peroxidase activity. Nonspecific binding were blocked in 10% goat serum in PBS at room temperature for 1 hour, then slides were incubated at 4 °C overnight with primary antibodies: α-SMA (Abcam, ab5694, 1:400), Mac3 (BD Pharmingen, 550292, 1:300), H60b (Santa Cruz Biotechnology, sc-20330, 1:100), H3K9Ac (Abcam, ab4441, 1:200), H3K56Ac (Epigentek, A-4026, 1:200). The slides were incubated with biotinylated secondary antibodies at 37 °C for 30 minutes and subsequently with horseradish peroxidase-labeled streptavidin solution at 37 °C for 20 minutes. Slides were then stained with diaminobenzidine (DAB kit, Vector Laboratories) and counterstained with hematoxylin.

Cells were collected, fixed and cell nuclei were isolated and sonicated. After pre-clearing, the supernatant were immunoprecipitated using normal rabbit IgG (Santa Cruz Biotechnology, sc-2027), Sirt6 antibody (Abcam, ab62739), H3K9Ac antibody (Abcam, ab4441), or H3K56Ac antibody (Epigentek, A-4026). The immunoprecipitated DNA were PCR amplified using indicated primers in Supplementary Table 7.

The antibodies used were as follows: Sirt6 (Abcam, ab62739, 1:1000; CST, 2590, 1:1000), H60b (Santa Cruz Biotechnology, sc-20330, 1:300), β-actin (Sigma, A5376, 1:5000), H3 (Abcam, ab1791, 1:1000), and H3K9Ac (Abcam, ab4441, 1:1000), H3K56Ac (Epigentek, A-4026, 1:1000), H3K14Ac (Abcam, ab52946, 1:1000), MICA/B antibody (R&D, MAB13001, 1:500).