Cuznsod

Equal amounts of protein from brain, spinal cord, gastrocnemius and quadriceps muscle were resolved by SDS-PAGE and transferred to PVDF membrane. The membranes were blocked and probed with CuZnSOD (Enzo Life Sciences, Inc., Farmingdale, NY, USA) and GAPDH (Sigma, St. Louis, MO, USA) antibodies overnight at 4 °C. Membranes were washed extensively and incubated with secondary antibodies linked to horseradish peroxidase (Santa Cruz Biotechnology, Dallas, TX, USA). Proteins were visualized using enhanced chemiluminescence reagent and signal intensities were quantified using ImageJ 1.45b software (developed by Wayne Rasband, National Institute of Health, Bethesda, MD).

Muscles were ground in a mortar and pestle under liquid nitrogen, and frozen muscle powder was placed into Radioimmunoprecipitation assay (RIPA) buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, and protease inhibitors. Samples were homogenized on ice and centrifuged at 10 000 g for 10 min at 4°C. Protein content of samples was determined using the bicinchoninic acid method (Sigma‐Aldrich, Poole, UK). For assessment of specific proteins in muscle, 20 mg of total protein was applied to a 4–20% mini‐PROTEAN TGX precast gel with a 4% stacking gel (Bio‐Rad Laboratories Ltd, Hemel Hempstead, UK). The separated proteins were transferred onto nitrocellulose membranes by western blotting. Membranes were probed using antibodies against calstabin, serca1, calsequestrin (Abcam, Cambridge, UK), DHPR, RYR (Thermo Scientific, USA), calcineurin, nuclear factor of activated T‐cells (NFAT), calpain (Cell Signaling, Hitchin, UK), and CuZnSod (Enzo, Farmingdale, NY, USA). Horseradish peroxidase conjugated anti‐rabbit IgG or anti‐mouse IgG (Cell Signaling) was used as secondary antibody. Peroxidase activity was detected using an ECL Plus substrate (Amersham International Cardiff, UK), and band intensities were analysed using Quantity One Software (Bio‐Rad Laboratories Ltd). All protein contents were normalized to protein levels determined by the ponceau stain.