Immobilon polyvinylidene diflouride membranes

Right-atrial tissue samples of about 20 mg were pulverized in liquid nitrogen and ‘homogenized’ in 200 µL of ice-cold lysis buffer containing 50 mM HEPES pH 7.4, 0.1% (v/v) Tween 20, 100 mM NaCl, 2.5 mM EGTA, 10 mM glycerol-2-phosphate, 10% (v/v) glycerol, and 1 mM DTT supplemented with a cocktail of protease inhibitors (Roche). Proteins were separated by SDS–PAGE (10% acrylamide: bisacrylamide) and electrotransferred onto Immobilon polyvinylidene diflouride membranes (Millipore). Membranes were incubated with primary and secondary antibodies diluted in 5% non-fat dry milk except for DHPR blots, for which SuperBlock™ Blocking Buffer (Thermo Scientific) was used. Antibodies against SERCA (#9580, Cell Signaling Technology), calsequestrin-2 (ab3516, Abcam), DHPR (ab81980, Abcam), and NCX1 (ab135735, Abcam) were used. After a standard washing protocol, detection was performed using the appropriate horseradish peroxidase-labelled IgG and the Supersignal™ detection system (Supersignal West Dura™, Pierce). Molecular-mass standards (Bioline) were used to estimate protein size and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; MAB374, Millipore) was used as a loading control. Immunoblots were digitized (GS-800 Calibrated Densitometer; Bio-Rad) and analysed with the Quantity One 4.6.3 software (Bio-Rad).