Tris glycine polyacrylamide precast gels

Samples were loaded into 4–20% Tris–Glycine polyacrylamide precast gels (ThermoFisher Scientific, Waltham, MA, USA) and electrophoresis was carried out. The electrophoresis under denaturing conditions (sodium dodecylsulphate polyacrylamide gel electrophoresis, SDS-PAGE) resolved proteins were transferred to iBlot stacks with regular polyvinylidene fluoride (PVDF) membranes using the iBlot 2 dry blotting system (ThermoFisher Scientific, Waltham, MA USA). Nonspecific binding sites were blocked with 5% nonfat dry milk in phosphate-buffered saline with tween (PBS-T) (in mmol/L, 3 KH₂PO₄, 10 Na₂HPO₄, 150 NaCl, and 0.15% Tween 20, pH 7.2–7.4) for 30 min at room temperature. Membranes were then incubated with specific primary antibodies for SERCA2a (1:500; ThermoFisher) and PLN (1:5000; Badrilla, UK), diluted in 5% bovine serum albumin in PBS-T overnight at 4 °C. After washing three times for 10 min, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies, diluted in 5% bovine serum albumin in PBS-T. After washing three times for 10 min, protein-antibody reactions were detected using Pierce SuperSignal Chemiluminescent Substrates (ThermoFisher Scientific, Waltham, MA USA). We performed detection and quantification of protein bands with a Bio-Rad ChemiDoc system and Image Lab software 5 (Bio-Rad, Hercules, CA USA).

Cells from the 96 well plates were collected using 2 × Laemmli sample buffer (Bio-Rad, Hercules, CA USA), 30 µL per well, and stored at − 20 °C. Samples were loaded into 4–20% Tris–Glycine polyacrylamide precast gels (ThermoFisher Scientific, Waltham, MA USA) and electrophoresis was carried out. The SDS-PAGE resolved proteins were transferred to iBlot stacks with regular PVDF membranes using the iBlot 2 Dry Blotting System (ThermoFisher Scientific, Waltham, MA USA). Nonspecific binding sites were blocked with 5% nonfat dry milk in PBS-T (in mmol/L, 3 KH₂PO₄, 10 Na₂HPO₄, 150 NaCl, 0.15% Tween 20, pH 7.2–7.4) for 30 min at room temperature. Membranes were then incubated with specific primary antibodies (Supplementary Table S1) diluted in 5% bovine serum albumin in PBS-T overnight at 4 °C. After washing 3 times for 10 min, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Supplementary Table S1) diluted in 5% bovine serum albumin in PBS-T. After washing 3 times for 10 min, protein-antibody reactions were detected using Pierce SuperSignal Chemiluminescent Substrates (ThermoFisher Scientific, Waltham, MA USA). Detection and quantification of protein bands were performed with a Bio-Rad ChemiDoc system and Image Lab software 5 (Bio-Rad, Hercules, CA USA).