Orai1

Protein samples (60 μg) were heated to 95 °C for 5 min and loaded onto 10% sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes and blocked with 5% nonfat dry milk for 1 h at room temperature. Membranes were washed with 0.1% PBST (phosphate-buffered saline (PBS) and Tween20) three times and then incubated with primary antibodies overnight at 4 °C. Antibodies against pERK1/2 (Cell Signaling, Beverly, MA), STIM1 (Cell Signaling, Beverly, MA), and ORAI1 (GeneTex, Hsinchu, Taiwan) were diluted 1: 2000, whereas the antibody against β-actin was diluted 1: 10000. Membranes were then washed with 0.1% PBST three times and incubated with a 1: 5000 dilution of anti-mouse or anti-rabbit HRP-conjugated immunoglobulin G (IgG, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. After washing with 0.1% PBST three times, signals were detected by an ECL-plus Western blotting detection system (Millipore).