Ecl detection kit

Cells were harvested and cellular proteins were extracted as mentioned above. Total cellular proteins from different time points were separated by SDS-PAGE and electrotransferred to nitrocellulose membranes (Whatman). The membranes were blocked with 5% non-fat milk powder dissolved in tris-buffered saline, containing 0.05% Tween-20, for 2 h at room temperature. Primary antibodies (see below) were diluted 1:500 in blocking buffer and incubated with the membranes overnight at 4 °C. Next, the membranes were incubated with the appropriate secondary antibodies diluted in blocking buffer at 1:3000 for 2 h at room temperature. Detected proteins were revealed using the ECL Detection Kit (KeyGen Biotech).
The following rabbit antibodies were used: Ribosomal Protein S4X Polyclonal Antibody (YT4135), Ribosomal Protein L17 Polyclonal Antibody (YT4098), QM Polyclonal Antibody (YT3916), Histone H3.1 Polyclonal Antibody (YT2163), Laminin γ-1 Polyclonal Antibody (YT2531), GNL3L Polyclonal Antibody (YT1938), Ribosomal Protein L7 Polyclonal Antibody (YT4118), and β-Actin Polyclonal Antibody YT0099). All the rabbit antibodies were purchased from ImmunoWay Biotechnology Company. The Peroxidase AffiniPure goat Anti-Rabbit IgG was used as the secondary antibody (Jackson ImmunoResearch Lab.).

Total protein was extracted using a protein extraction kit (KeyGEN BioTECH, Nanjing, China) to measure the expression of apoptosis-related factors. Protein concentrations were measured using the BCA kit (Sigma–Aldrich). Next, the proteins (30 μg) were separated using 10% SDS-PAGE and then transferred onto PVDF membranes (Merck Millipore, Billerica, MA, USA). After blocking with 5% skimmed milk, the membranes were incubated with primary antibodies at 4°C overnight, followed by incubation with secondary antibodies at room temperature for 1 h. Protein bands were developed using the ECL detection kit (KeyGEN BioTECH) and images were obtained. GAPDH was used as an internal control. The antibodies (Cell Signaling Technology, Danvers, MA, USA) used in this study were shown as follows: anti-XIAP (#2042, 1:1000), Caspase-3 (#9662, 1:1000), Bax (#2774, 1:1000), Bcl-2 (#3498, 1:1000) GAPDH (#2118, 1:1000), and HRP linked anti-Rabbit (#7074, 1:3000).

The total protein from the tissue and H9C2 cardiomyocytes was extracted using a cell lysis solution (Beyotime Institute of Biotechnology) and phosphatase inhibitor (Meilun Bio, Dalian, USA) and quantified using the Bicinchoninic Acid (BCA) Protein Assay Kit (Beyotime). An equal amount of protein (40 mg/well) was separated by 10% and 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes for 1.5 hours at 100 V. The membrane was then blocked with 5% BSA at room temperature for 1.5 hours. GAPDH, akt2, p-akt2, cleaved caspase3, bax, and bcl-2 primary antibodies were incubated with the membranes at 4°C overnight (Akt2; 1 : 200; Santa Cruz Biotechnology, Santa Cruz, CA, USA; p-akt2; 1 : 500; Abcam, Cambridge, MA, USA; cleaved caspase3; 1 : 500; Cell Signaling Technology, Danvers, MA, USA; bax and bcl-2; 1 : 500; Proteintech; GAPDH; 1 : 10000; Abways). Subsequently, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Proteintech) at room temperature for 2 hours. The blotting was visualized using enhanced chemiluminescence (ECL detection kit, KeyGEN Biotech, Jiangsu, China) on the c300 Chemiluminescent Western Blot Imaging System (Azure Biosystems, Dublin, GA, USA).