Anti ryr2

After isolation of S2814A and WT cardiomyocytes (see above), cells were either incubated in NT or ISO solution (100 nmol·L⁻¹) for 10 min. Cells were homogenized in Tris buffer containing (in mm): 20 Tris/HCl, 200 NaCl, 20 NaF, 1 Na₃VO₄, 1 DTT, 1% Triton X‐100 (pH 7.4), complete protease inhibitor cocktail (Roche diagnostics, Mannheim, Germany) and phosphatase‐inhibitor mixture (PhosSTOP; Roche). Protein concentration was determined by BCA assay (Sigma‐Aldrich Co., St. Louis, MO, USA). Denatured proteins were separated on SDS‐polyacrylamide gels (5–12%) and transferred to nitrocellulose membranes (GE Health Care, Chalfont St Giles, UK). Specific proteins were detected using anti‐RyR2 (1 : 10 000, Sigma‐Aldrich Co.), anti‐pS2814‐RyR2 (1 : 1000; Badrilla, Leeds, UK), anti‐pS2808‐RyR2 (1 : 1000; Badrilla), antibodies followed by HRP‐conjugated donkey anti‐rabbit IgG antibodies (GE Health Care). Chemiluminescent detection was performed with WesternBright™ Chemiluminescent Substrate (Biozym Scientific GmbH, Hess. Oldendorff, Germany). Phosphorylation levels of the proteins were normalized to total protein expression.

Protein extraction and immunoblotting were performed as previously described²⁶ . Briefly, membranes were incubated with the following antibodies overnight at 4 °C: anti-GAPDH (1:1000; Santa Cruz), anti-P-PLN-Thr17, anti-PLN, anti-P-RyR2-Ser2814 (1:5000; Badrilla), anti-RyR2 (1:2000; Sigma-Aldrich). Full blots are shown in the Supplemental Material section.

Frozen LV was homogenized in RIPA buffer (Millipore, Schwalbach am Taunus, Germany) containing protease and phosphatase inhibitor cocktail tablets (Roche, Mannheim, Germany). Extracted proteins (20 µg) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a nitrocellulose or Polyvinylidene Difluoride (PVDF) membranes (Bio-Rad, München, Germany). Membranes were blocked for 1 h with 5% milk in TBS-Tween at room temperature and then incubated with the following primary antibodies: Rabbit polyclonal anti-phospho-Ser2814-RyR2 (Badrilla, Leeds, UK), anti-phospho-Thr-17 Phospholamban (Badrilla), anti-RyR2 (Sigma-Aldrich, Munich, Germany), anti-CaMKIIγ and anti-CaMKIIδ (Thermo Fisher, Bonn, Germany), and mouse monoclonal anti-phospho-CaMKII (Affinity BioReagents, Golden, CO, USA), anti-Phospholamban (Millipore, Hamburg, Germany), anti-Serca2a (Affinity BioReagents), and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Blots were subsequently incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, and finally were detected using an enhanced chemiluminescent detection system (Amersham Bioscience, Braunschweig, Germany) according to the manufacturer’s instructions. For quantification, band intensity was normalized to total protein load obtained from Ponceau-stained blot using Image Lab software (Bio-Rad).