Protein Expression Analysis of Frozen RV

Frozen RV was homogenized using ice-cold lysis buffer and proteinase inhibitor cocktail (Manchini et al., 2014 (link)). Lysates corresponding to 30 μg of protein were subjected to 10% SDS-PAGE. Separated proteins were transferred to PVDF membrane (Amersham Biosciences, NJ, USA) and transfer effectiveness was examined with 0.5% Ponceau S. After blocking with 5% non-fat dry milk for 2 h at room temperature, PVDF membranes were probed with Abcam (Cambridge, MA, USA) primary antibodies for rabbit ant-Akt₁ (1:5000), rabbit anti-p-Akt₁ (1:2500), rabbit anti-Caspase3; rabbit anti-Bax (1:1000), rabbit anti-Bcl-2 (1:1000), rabbit anti-Bcl-xL (1:500), rabbit anti-β-MHC (1:5000), rabbit anti-α-MHC (1:5000), rabbit anti-L-type Ca⁺⁺ (1:500), rabbit anti-ryanodine receptor (1:1000), rabbit anti-Serca 2 (1:1000), and rabbit anti-Na⁺/Ca⁺⁺ exchanger (1:100) in overnight incubation. Membranes were then washed five times with PBS and incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit (1:20,000; Zymed, CA, USA). Membranes were again washed five times with blocking buffer and then rinsed twice with PBS. Antibodies binding were detected by chemiluminescence reagents (Amersham Biosciences, NJ, USA), and images were captured using an Amersham Imager 600 system. Quantification of target proteins was normalized for the internal control glyceraldehyde 3-phosphate dehydrogenase.