Ab191385

ChIP assay was performed using Pierce Magnetic ChIP kit (Pierce). In brief, HAPI cells and BMVECs were crosslinked with 1% formaldehyde and harvested. Chromatin fragments were prepared by MNase digestion, and incubated with anti-SIRT6 (ab191385; 3 μg, Abcam), anti-H3 (ab1791; 2 μg, Abcam), anti-H3K9ac (#9649; 10 μl; CST), anti-H3K56ac (ab195478; 5 μg, Abcam) antibody or corresponding normal IgG. DNA was further purified and analyzed by qRT-PCR. Amplification conditions were as follows: 95 ˚C for 1 min, 40 cycles consisting of 95 ˚C for 30 s, 65 ˚C for 30 s and 72 ˚C for 30 s, and 72˚C for 5 min. The following primers were used: Forward 5’-GGCACAACCAGCTGGTTGAA-3’; Reverse 5’-TGAGCCGAGTGGGTTCAAGA-3’.

After a 30-min blocking with BSA, slices of the aorta were treated at 4 °C for an entire night with mouse monoclonal anti-Sirt6 (ab191385, 1:100; Abcam), α-smooth muscle actin (SMA) (GB13044, 1:200; Servicebio), and anti-CD68 (GB11067, 1:100; Servicebio). Thereafter, rabbit anti-mouse secondary antibody (1:1,000; Abcam) was used to stain the sections for 1 h at room temperature (RT) before rinsing and mounting them with a medium containing 4,6-diamidino-2-phenylindole (DAPI). Haematoxylin was used to stain the nucleus. Subsequently, an upright fluorescent microscope and an Olympus FV1000 laser confocal microscope were used for the purpose of visualising the sections.

Cells or tissue homogenates were added with RIPA lysis buffer on ice for 15 min. The lysates were collected and centrifuged at 13000 rpm at 4°C for 20 min. Proteins were separated on sodium dodecyl sulfate polyacrylamide gel following with transferred to PVDF membranes (Millipore). The membranes were blocked with 5% skim milk and incubated with specified antibodies. Band intensity was analyzed with the ImageJ software.
Antibodies against α-SMA (ab21027), SIRT6 (ab191385) and HIPK2 (ab108543) were obtained from Abcam. Antibody against Tubulin (11224-1-AP) was obtained from Proteintech. Antibody against HIPK2 (sc-100383) was obtained from Santa Cruz Biotechnology. Antibodies against E-cadherin (3195S), collagen I (72026S), GAPDH (5174S), secondary anti-rabbit (4412S) and anti-mouse (4408S) were purchased from Cell Signaling Technology.

Proteins were isolated using RIPA lysate buffer. Protein concentrations were determined by the BCA protein assay kit (Beyotime, Shanghai, China). Protein samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% BSA for 1 h at room temperature, the membranes were incubated with primary antibodies overnight at 4 °C and then incubated with the secondary antibodies for 1 h at room temperature. Finally, the immunoblot was visualized by a gel densitometric scanning system and quantified using ImageJ (National Institutes of Mental Health, Bethesda, MD, USA).
The primary antibodies were as follows: SIRT6 (#ab191385; abcam; 1:1000), H3K9Ac (#ab32129; abcam; 1:1000), ERK1/2 (#4695S; CST; 1:1000), p-EKR1/2 (#9101S; CST; 1:1000), acetylated-lysine antibody (#9441S; CST; 1:1000), ZO-1 (#61-7300; Invitrogen; 1:500 and #ab276131; abcam; 1:1000), occludin (#ab216327; abcam; 1:1000), GAPDH (#A19056; Abclonal; 1:2000), LC3B (#ab51520; abcam; 1:1000), Beclin1 (#A21191; Abclonal; 1:1000), and α-tubulin (#AF0001; Beyotime; 1:1000).

Aortic tissue was processed for protein extraction, and macrophages were grown as directed by the manufacturer (Invitrogen, Carlsbad, CA, USA) [22 (link)] and quantified using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Subsequently, proteins were isolated via SDS-PAGE using antibodies against Beclin1 (ab62472; Abcam), SNF2H (ab72499; Abcam), P62 (ab91526; Abcam), LC3A/B (ab128025; Abcam), Wnt1 (ab15251; Abcam), Sirt6 (ab191385; Abcam), β-catenin (ab32572; Abcam), GAPDH (ab181602; Abcam), LAMP1 (ab25245; Abcam), PLIN2 (ab108323; Abcam) and Adipophilin (ab108323; Abcam). A chemiluminescence system (Amersham Bioscience, Buckinghamshire, UK) was used to visualize the blots, and the Image-Pro Plus program (Media Cybernetics, MD Rockville, USA) was employed for quantifying the images.

Co‐immunoprecipitation (Co‐IP) assay was performed by following the methods indicated in a study.¹⁹ The primary antibody SIRT1 (1:200; ab32441, Abcam) and secondary antibodies Pcsk9 (1:50, ab191385, Abcam) and anti‐acetyl Lysine (ab21623, 1:50, Abcam) were used.

HUVECs were initially lysed with RIPA lysis buffer (Beyotime) with phenylmethanesulfonyl fluoride (PMSF; Beyotime) and total cellular proteins were harvested. Protein extracts were collected and quantified by the BCA protein assay kit (BioRad, Hercules, CA, USA). Notably, equal amounts (30 μg) of protein samples in each group were subjected to SDS-PAGE. Proteins were followingly transferred to PVDF membranes (Millipore), which were instantly blocked with 5% skimmed milk for 1 h and subsequently incubated with primary antibodies anti-SIRT6 (Abcam, ab191385, 1 : 1000) and anti-GADPH (Abcam, ab9485, 1 : 1000) at 4°C overnight. The membranes were then immersed in Tris-buffered saline with Tween-20 (TBST), incubated with secondary antibody (Abcam, ab97051, 1 : 5000) for 2 h and rinsed with TBST again. Ultimately, the protein signal was developed by the ECL Plus assay kit (Pierce, Rockford, IL, USA).