Pierce ecl western blotting substrate

Cardiac tissues were lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology). The protein concentration was determined using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Then, 20 ug of protein was separated via 12% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were first blocked with 5% non-fat milk for 1 h at RT, and then incubated with specific primary antibodies overnight at 4°C, including anti-TLR4 (1:1,000; cat. no. ab13556; Abcam), anti-TLR2 (1:2,000; cat. no. sc-10739; Santa Cruz Biotechnology, Inc.), anti-RP105 (1:1,000; cat. no. Sc-13592; Santa Cruz Biotechnology, Inc.), anti-Bcl-2 (1:1,000; cat. no. ab196495; Abcam), anti-cleaved caspase-3 (1:1,000; cat. no. ab2302; Abcam), anti-MYD88 (1:1,000; cat. no. ab2064; Abcam) and anti-NF-κB antibody (1:1,000; cat. no. Ab32536; Abcam). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000; cat. nos. Ab97046 and Ab97057; Abcam) at RT for 1 h. The bands were exposed with Pierce ECL western blotting substrate (cat. no. 32106; Thermo Fisher Scientific, Inc). The expression of GAPDH (1:3,000; cat. no. Ab181602; Abcam) was detected to normalize the signals. The expression of proteins was qualified using ImageJ software (1.47; National Institutes of Health)

Protein was extracted from the whole-cell lysate using RIPA buffer (Cell Signaling Technology, Inc.). Subsequently, western blotting was performed using a routine method. Briefly, the protein concentration was measured with a BCA assay kit (Bio-Rad Laboratories, Inc.), and 50 µg/lane of protein was resolved by a 10% Mini-PROTEAN TG gel (Bio-Rad Laboratories, Inc.) and loaded onto a PVDF membrane. The membranes were blocked using a Tris buffer containing 5% skim milk and 0.1% Tween-20 for 60 min at room temperature. Subsequently, the membrane was incubated overnight at room temperature with anti-PDCD6 (1:1,000; cat no. ab109181; Abcam) and anti-PETN (1:1,000; cat no. ab267787; Abcam) primary antibodies, washed with a Tris buffer containing 0.1% Tween-20 three times and incubated with an HRP-conjugated secondary antibody (1:2,000; cat no. ab6721; Abcam) at room temperature for 1 h. The gel was visualized using Pierce™ ECL Western Blotting Substrate (cat. no. 32109; Thermo Fisher Scientific, Inc.) and analysed by ImageJ 1.44e software (National Institutes of Health). Anti-β-actin (1:1,000; cat. no. MA5-15739; Invitrogen; Thermo Fisher Scientific, Inc.) was used as the internal control to calculate the relative expression levels of PDCD6 and PTEN proteins.

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.).