Quantity one version 3

Following euthanasia, the heart, aortic trunk and right apex of the left ventricular myocardium were dissected from the mice. The tissues were rinsed with pre-cooled 0.9% saline, fixed in 4% paraformaldehyde for 2 h at room temperature and embedded in paraffin. Paraffin-embedded tissue samples were cut into 5-µm serial sections. The tissue sections were subsequently deparaffinized in xylene at room temperature and rehydrated in a descending ethanol series. Sections were then blocked with 3% hydrogen peroxide for 10 min at 25˚C to inhibit endogenous peroxidase activity. Tissue sections were incubated with the following primary antibodies for 12 h at 4˚C: Anti-MMP-9 (1:4,000; cat. no. ab38898; Abcam) and anti-CD40L (1:4,000; cat. no. ab2391; Abcam). Following the primary antibody incubation, the sections were incubated with an Alexa Fluor 488-conjugated secondary antibody (1:2,000; ab150077; Abcam) at 25˚C for 2 h. The slides were observed under an Olympus IX73 microscope (magnification, x100; Olympus Corporation). The quantification of expression levels was performed using Quantity One version 3.2 software (Bio-Rad Laboratories, Inc.).

Total protein was extracted from arterial tissue (1.0 g) using RIPA lysis buffer (Thermo Fisher Scientific, Inc.). Total protein was quantified using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Inc.) and 40 µg protein/lane was separated using 15% SDS-PAGE. The separated proteins were subsequently transferred onto a nitrocellulose membrane and blocked with 5% BSA (Sigma-Aldrich; Merck KGaA) for 1 h at 37˚C. The membranes were subsequently incubated with the following primary antibodies for 12 h at 4˚C: Anti-PI3K (1:2,000; cat. no. ab232997; Abcam), anti-phosphorylated (p)-PI3K (1:1,000; cat. no. ab182651; Abcam), anti-AKT (1:2,000; cat. no. ab185633; Abcam), anti-p-AKT (1:2,000; cat. no. ab133458; Abcam), anti-mTOR (1:2,000; cat. no. ab32028; Abcam) and anti-β-actin (1:1,000; cat. no. ab8226; Abcam). Following the primary antibody incubation, membranes were incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5,000; ab6721; Abcam) for 24 h at 4˚C. Protein bands were visualized using the Pierce^™ ECL Western Blotting Substrate (cat. no. 32209; Invitrogen; Thermo Fisher Scientific, Inc.). Protein expression was quantified using Quantity-One version 3.2 software (Bio-Rad Laboratories, Inc.).