Western blotting

Western blot analyses were carried out to measure the expression levels of multiple proteins in H9c2 cells. The protocol was performed as described (22). Briefly, extracted proteins were separated in 10% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were blocked with 5% (w/v) nonfat dry milk in Tris-buffered saline for 2 h at room temperature, and incubated with the respective primary antibodies at 4°C overnight. The antibodies used were as follows: phospho-MAPK, total-MAPK, (phospho cat. no. 9910; 1:1,000; total cat. no. 9926, 1:1,000), poly-ADP-ribose polymerase (PARP; cat. no. 9532; 1:1,000), B-cell lymphoma 2 (Bcl-2; cat. no. 3498; 1:1,000 (23) (all from Cell Signaling Technology, Inc.); PAR2 (cat. no. 180953, 1:1,000; Abcam). After incubation with horseradish peroxidase-conjugated secondary antibodies Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (product code no. 111-035-003; 1:5,000; Jackson ImmunoResearch Laboratories, Inc.) at room temperature for 2 h. GAPDH was evaluated as a loading control. Antibody-bound bands were detected with an enhanced chemiluminescence (ECL) detection kit (EMD Millipore). Quantification was performed using Quantity One 4.4.0 software (Bio-Rad Laboratories, Inc.).