Ab14348

Cellular and tissue proteins were extracted with lysis buffer, which contained 1% (vol/vol) Nonidet P-40, 150 mmol/L NaCl, 20 mmol/L Tris-HCl, pH 8.0, with the addition of Roche protease inhibitor cocktail tablets (Sigma–Aldrich, St. Louis, MO, USA, 04693132001). The protein concentrations were determined with the DC Protein Assay (Bio-Rad, Hercules, CA, USA, 500-0002). The proteins were separated in 12% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% BSA in TBS for 2 h at room temperature, the membranes were incubated with primary antibodies at 4°C overnight. The primary antibody was anti-GAPDH (1:5000, Abcam, Cambridge, UK, ab8245), anti-Orai1 (1:1000, Sigma–Aldrich, St. Louis, MO, USA, O8264), anti-STIM1 (1:1000, Cell Signaling, Danvers, MA, USA 5668S), anti-ANF (1:3000, Abcam, ab14348), or anti-cTnT (1:3000, Abcam, ab8295). Immunodetection was accomplished using horseradish peroxidase-conjugated secondary antibody, followed by analysis via the ECL detection system. The intensity of immunoblot bands was quantified using ImageJ Software.

Total protein was extracted with lysis buffer having 1% (vol/vol) Nonidet P-40, 150 mmol/L NaCl, 20 mmol/L Tris-HCl (pH 7.4), and Roche protease inhibitor cocktail (Sigma–Aldrich, St. Louis, MO, United States, 04693132001). The sample protein concentrations were determined using the DC Protein Assay (Bio-Rad, Hercules, CA, United States, 500–0002). The protein samples were resolved by 12% SDS-PAGE and then transferred onto PVDF membranes. The membranes were blocked with 5% BSA in TBS for 2 h at room temperature (RT), and then overnight incubated with primary antibodies at 4°C. The used primary antibodies were as follows: anti-GAPDH (1:6000, Abcam, Cambridge, United Kingdom, ab8245), anti-Orai1 (1:1000, Sigma–Aldrich, St. Louis, MO, United States, O8264), anti-STIM1 (1:1000, Abcam, ab108994), anti-ANF (1:1000, Abcam, ab14348), anti-β-Actin (1:1000, Abcam, ab14348), and anti-LC3 (1:1000, SIGMA, ab8295). Immunoreactive bands were visualized using the horseradish peroxidase-conjugated secondary antibodies. The intensity of immunoblotted bands was quantified using the ImageJ software.