Sc 7294

RAW 264.7 cells were lysed by radioimmunoprecipitation assay buffer with protease inhibitor cocktail (Cell Signaling Technology) at 0–4°C. The protein concentrations for each sample were measured, and 20 µg protein lysates from each sample were subjected to 8 or 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis, and the separated proteins were transferred onto polyvinylidene difluoride membranes. These blots were blocked with Tris-buffered saline containing 0.05% Tween-20 (TBST), supplemented with 5% non-fat dry milk for 1 h at room temperature, and incubated with primary antibodies (mouse nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) antibody (1:2000, sc-7294, Santa Cruz Biotechnology), rabbit TRAP antibody (1:10000, ab191406, Abcam), mouse cathepsin K antibody (1:1000, sc-48353, Santa Cruz Biotechnology), mouse Cyclin D1 antibody (1:1000, sc-8396, Santa Cruz Biotechnology), and mouse α Tubulin antibody (1:10000, sc-23948, Santa Cruz Biotechnology)) overnight at 4˚C. After the incubation with primary antibodies, the membranes were washed with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Peroxidase labeling was detected with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific). The relative expression levels of proteins for western blotting were normalized to α Tubulin.

Samples for Western blotting were collected and stored in T-PER (Thermo Fisher Scientific) at −80 °C until use. The samples were homogenized in ice-cold T-PER with Cell Destroyer (Bio Medical Science), and the total protein concentration was determined using BCA protein assays (Thermo Fisher Scientific). Equal amounts of protein (3 to 5 µg of protein sample per well) were loaded on 10% sodium dodecyl sulfate–polyacrylamide electrophoresis gels (SuperSep Ace) and transblotted to polyvinylidene fluoride membranes. The membranes were incubated in blocking buffer (Blocking One, nacalai tesque) for 1 h and then overnight with primary antibodies, including mouse anti-NFATc1 at 1:5,000 dilution (sc-7294, Santa Cruz); rabbit anticalcineurin at 1:5,000 dilution (GTX59619, CST); mouse anti-MyoD at 1:1,000 dilution (sc-377460, Santa Cruz); guinea pig anti-p62/SQSTM1 at 1:5,000 dilution (GP62-C, PROGEN); and mouse antiβ-actin at 1:5,000 dilution (A5441, Sigma-Aldrich). Primary antibodies were detected by incubation with the appropriate secondary antibody for 1 h at room temperature and then with the ECL Prime Western Blotting Substrate (GE Healthcare) for 5 min. Protein expression was measured using WSE-6100LuminoGraph I (ATTO). The densities were measured using ImageJ software (NIH).