H9c2 cardiomyocytes from each group were collected and total protein was extracted after cell lysis in RIPA buffer (Protech Technology Enterprise Co., Ltd.) at 4˚C for 20 min, followed by centrifugation at 8,798 x g for 10 min at 4˚C. Protein concentration was measured using a BCA kit (cat. no. A045-4-2; Nanjing Jiancheng Bioengineering Institute). Total protein extract from each group (20 µg/lane) was separated by SDS-PAGE on a 10% gel and transferred onto PVDF membranes (EMD Millipore). Following blocking with 5% skimmed milk for 2 h at room temperature, membranes were incubated with the following primary antibodies at 4˚C overnight: Anti-B-cell lymphoma 2 (Bcl-2; cat. no. ab196495; 1:1,000); anti-Bcl-2-associated X protein (Bax; cat. no. ab32503; 1:1,000); anti-poly(ADP-ribose) polymerase (PARP; cat. no. ab191217; 1:1,000); anti-cleaved PARP (cat. no. ab32064; 1:1,000); anti-Nrf2 (cat. no. ab92946; 1:1,000) and anti-HO-1 (cat. no. ab189491; 1:2,000; all Abcam); anti-phosphorylated (p)-NF-κB (cat. no. 3033; 1:1,000) and anti-NF-κB (cat. no. 8242; 1:1,000; both Cell Signaling Technology, Inc.) and β-actin (cat. no. ab8227; 1:1,000; Abcam). The membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibody (cat. no. ab6721; 1:2,000; Abcam) for 1 h at room temperature. The protein bands were visualized using ECL Western Blotting Substrate (Pierce; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Densitometry analysis was performed using ImageJ 1.52a software (National Institutes of Health) with β-actin as the loading control.