Supersignal west dura maximum sensitivity substrate

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed by loading 50 µg of total protein lysate (without nuclei) extracted from HEK-293 cells on 12% polyacrylamide gels. Following electrophoresis, separated proteins were transferred to methanol-activated polyvinylidene fluoride (PVDF) membranes with the Pierce G2 Fast Blotter (Thermo Fischer Scientific). Membranes were incubated in 5% nonfat milk in TBS-T buffer (15 mM Tris-HCl, 140 mM NaCl, 0.05% Tween20^®, pH 7.4) for 1 h at room temperature to block nonspecific protein binding. Subsequently, the membranes were incubated with primary antibodies overnight at 4 °C. The primary antibodies were anti-ORAI1 (Alomone, Jerusalemn, Israel: Acc-060), used at a 1/200 dilution, and anti-β-actin Sigma-Aldrich, St. Quentin Fallavier, France: A5441), used at a 1/5000 dilution. Following washes in TBS-T, the membranes were incubated for 1 h with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibodies diluted at 1/100,000 (Chemicon). Chemiluminescent detection of bound secondary antibodies was captured with Amersham Imager 600 (GE Healthcare Life Sciences) using the SuperSignal™ West Femto Maximum Sensitivity Substrate and the SuperSignal™ West Dura Maximum Sensitivity Substrate (Thermo Fischer Scientific, Illkirch, France).