Detection of protein expression and phosphorylation levels by Western blotting

For Western blot analysis, the cardiomyocytes were treated with vehicle or ATX-II (1 nmol/L) and incubated for 15 min at room temperature. The harvested cardiomyocytes were frozen immediately. The proteins were denatured for 5 min at 95°C under non-reducing conditions for PKARIα dimer and PKARIα monomer. To assess pSer2809 RyR2, pSer2814 RyR2, RyR2, pSer16 phospholamban (PLB), and PLB, the proteins were denatured for 30 min at 37°C in presence of 10% β-mercaptoethanol. They were denatured for 5 min at 95°C in presence of 10% β-mercaptoethanol for assessment of pThr287 CaMKII and CaMKIIδ expression. Following denaturation, the proteins were separated on 5% (RyR2, pSer2809 RyR2, pSer2814 RyR2), 8% (PKARIα dimer, PKARIα monomer, pThr287 CaMKII, and CaMKIIδ), or 12.5% (PLB, pSer16 PLB) SDS-polyacrylamide gels, transferred to a nitrocellulose membrane and incubated with the following primary antibodies: rabbit polyclonal anti-pThr287 CaMKII (1:1,000, PhosphoSolutions, Aurora, CO, USA), rabbit polyclonal anti-CaMKIIδ (1:10.000, Thermo Fisher Scientific Inc. of Waltham, MA, USA), mouse monoclonal anti-PKARIα (1:1,000, BD Biosciences, Heidelberg, Germany), rabbit polyclonal anti-RyR2 antibody (1:10.000, Sigma-Aldrich, St. Louis, MO, USA), rabbit polyclonal anti-pSer2809 RyR2 antibody (1: 1,000, Badrilla, Leeds, United Kingdom), rabbit polyclonal anti-pSer2814 RyR2 antibody (1:1,000, Badrilla, Leeds, United Kingdom), mouse monoclonal anti-PLB (1:10.000, Thermo Fisher Scientific Inc. of Waltham, MA, USA), rabbit polyclonal anti-pSer16 PLB (1:500, Badrilla, Leeds, United Kingdom), and mouse monoclonal anti-GAPDH (1:10.000, Sigma-Aldrich, St. Louis, MO, USA) at 4°C overnight. Secondary antibodies were horseradish peroxidase (HRP)-conjugated sheep anti-mouse and donkey anti-rabbit IgG (1:10.000, GE Healthcare, Chicago, Illinois, USA) and incubated for 1 h at room temperature. For chemiluminescent detection, Immobilon™ Western Chemiluminescent HRP Substrate (Millipore) was used. The values were normalized to GAPDH and WT vehicle values later.