Pa5 29559

Total protein was extracted from the left ventricular myocardium utilizing Total Protein Extraction Kit (Solarbio). Protein samples were separated by 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel electrophoresis and transferred onto the nitrocellulose membranes. The membranes were incubated with the primary antibodies, anti-atrial natriuretic peptide (ANP) (1:1,000 dilution; PA5-29559, Thermo Fisher Scientific, Waltham, MA, USA), anti-β-myosin heavy chain (β-MHC) (1:1,000 dilution; ab37484, Abcam, Cambridge, MA, USA), anti-STIM1 (1:1,000 dilution; MA1-19451, Thermo Fisher Scientific, Waltham, MA, USA), anti-Orai1 (1:1,000 dilution; ab111960, Abcam, Cambridge, MA, USA) at 4 °C overnight after blocking with 5% skimmed milk. The membranes were stained with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (1:5,000; ab270144, Abcam, Cambridge, MA, USA) or HRP-conjugated anti-rat IgG antibody (1:5,000; ab7097, Abcam, Cambridge, MA, USA) at room temperature. GAPDH antibody (1:5,000; ab8245, Abcam, Cambridge, MA, USA) served as internal control. The bands were developed by enhanced chemiluminescence reagent (Yeasen, Shanghai, China), and the data were analyzed by Image J software. The resulting ratios, expressed as fold change, were used to compare relative protein levels across the samples on blots.