Immunoblot Analysis of Phosphorylated RyR2

Immunoblot analysis was performed as previously described³⁶ . Heart tissue was homogenized in ice-cold RIPA buffer containing protease inhibitor (cOmplete Protease Inhibitor Cocktail, Merck)) and phosphatase inhibitor (PhosSTOP, Sigma). Supernatant was collected after centrifugation at 15,000 × g and an aliquot containing equal amount of protein was mixed with loading buffer (125 mM Tris-HCl pH 6.8, 30% glycerol, 10% SDS, and 0.6 M DTT). Without heat denaturation, protein sample was subjected to SDS–PAGE and transferred to PVDF membrane (Thermo Fisher Scientific). Specific antibodies against phospho-RyR2(Ser2808) (1:1000 dilution), phospho-RyR2(Ser2814) (1:1000 dilution; Badrilla)^{36 –39 (link)}, β-actin (1:2500 dilution; Invitrogen)^{40 (link)–42 (link)} were used as primary antibodies. Horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:3000 dilution; Cell Signaling Technology) and anti-mouse IgG antibody (1:3000 dilution; Cell Signaling Technology) were used as secondary antibodies. Immunoreactive signals were detected with ECL prime Western Blotting Detection System (GE Healthcare).