Ab111960

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.

Cells were lysed on ice with RIPA buffer (P0013B, Beyotime, Shanghai, China), and the supernatant was collected after centrifugation. Then, 10% SDS-PAGE gel electrophoresis was carried out, followed by being transferred to a PVDF membrane. After a 2-h block in a western blotting buffer (5% non-fat milk), the membrane was incubated overnight in a shaker with primary antibody dilution. The primary antibodies used specifically were OPN (bs-23258R, rabbit, 1:1000, Bioss, Beijing, China; ab63856, rabbit, 1:1000, Abcam, Cambridge, UK), Orai1 (ab111960, rabbit, 1:1000, Abcam, Cambridge, UK; 28411-1-AP, rabbit, 1:500, Proteintech, Wuhan, China), STIM1 (11565-1-AP, rabbit, 1:1000, Proteintech, Wuhan, China), eNOS (bs-20608R, rabbit, 1:1000, Bioss, Beijing, China), p-eNOS (bs-3589R, rabbit, 1:1000, Bioss, Beijing, China), and GAPDH (TA-08, mouse, 1:2000, Zsbio, Beijing, China). Afterward, HRP-conjugated secondary antibodies were diluted in a secondary antibody dilution solution, specifically goat anti-rabbit (ZB-2301, Zsbio, Beijing, China) and goat anti-mouse (ZB-2305, Zsbio, Beijing, China) at a ratio of 1:20000. After 2 h of incubation at room temperature, an ECL detection kit (340958, Thermo, Massachusetts, USA) was used to detect the proteins. The density quantification of the immunoblot was carried out with a chemiluminescent gel imaging analysis system (Bio-Rad, California, USA).