Sc 7294

Cultured murine cells were lysed in RIPA buffer (Nacalai Tesque, Kyoto, Japan). Protein concentration was determined using a bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). The denatured lysate mixed with Novex™ Tris–Glycine SDS Sample Buffer (2×; Thermo Fisher Scientific) was loaded onto Novex™ WedgeWell™ 4–12% Tris–Glycine gels (Thermo Fisher Scientific) for electrophoresis. The gels were electroblotted onto a PVDF membrane using iBlot 2 PVDF mini stacks (Thermo Fisher Scientific). Membranes were blocked with Blocking One (Nacalai Tesque) for Nfatc1 detection and with 5% skim milk (FUJIFILM Wako Pure Chemical Corporation) for β-actin. The membranes were incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies at room temperature for 1 h. ECL Prime Western Blotting Detection Reagent (GE Healthcare Bioscience, Chicago, IL, USA) was added to the membranes, and the chemiluminescent signal was detected using a CCD camera (Vilber, Collégien, France). The primary antibodies used were mouse monoclonal antibodies against Nfatc1 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-7294, 1:1000 dilution) and β-actin (Sigma-Aldrich, A1978, 1:2000 dilution). The secondary antibody was an ECL peroxidase-labeled anti-mouse antibody (GE Healthcare Bioscience. 1:10,000 dilution).