Skf 96365

Proteins were separated by electrophoresis on 1D-PAGE 15% polyacrylamide gels. Gels were loaded with an equal volume on each lane. Phosphorylated proteins were detected by Pro-Q^® Diamond stain following the manufacturer’s protocol (Invitrogen, Waltham, MA, USA). In short, the gels were fixed in 5% acetic acid and 50% methanol and incubated with Pro-Q^® Diamond stock solution in 25 mL of staining buffer for 1.5 h. The gel was scanned using a Typhoon 9400 (GE Healthcare, Chicago, IL, USA). Subsequently, total proteins were detected by SYPRO^® Ruby stain. The gels were fixed with 50% methanol and 7% acetic acid and stained with SYPRO^® Ruby stain overnight. Phospho-bands were identified according to the molecular weight, normalized to the total lane individually, and then averaged (n = 5 per group). For positive control groups, the STIM1 inhibitor SKF 96365 (Santa Cruz, Dallas, TX, USA) was applied (5 µM) for 72 h, and the calcineurin inhibitor FK506 (Sigma-Aldrich, St. Louis, MO, USA) was applied (30 μM) for 24 h. The same Pro-Q method was applied to the mice atrial tissue (n = 3 per group).