Halts protease inhibitor

For the preparation of tissue extracts, approximately 100 mg of the left ventricle was pulverized in liquid nitrogen. The powder was subsequently resuspended in 1 mL ice-cold buffer containing 20 mM HEPES, pH 7.4, 2 mM EGTA, 50 mM β-glycerophosphate, 1 mM dithiothreitol, 1 mM Na₃VO₄, 10% glycerol, 1% Triton X-100 and Halts Protease Inhibitor (Thermo Scientific, Fair Lawn, NJ). Resuspended tissue was further homogenized using a mechanically driven Potter-Elvehjem glass-glass homogenizer with approximately 14 up and down strokes. The homogenates were subsequently rotated for 1 hour at 4 °C for efficient cell lysis. Finally, the homogenates were centrifuged at 10,000 g for 10 minutes at 4 °C and the supernatant was transferred to clean tubes. Protein concentrations were determined using Bradford Assay [25 (link)], and aliquots were stored at −80 °C until further analysis.