Horseradish peroxidase conjugated anti rabbit igg or anti mouse igg

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.

SW-480 cells were seeded in 6-well plates at density of 5.4× 10⁴ cells per well and incubated at 37°C in 5% CO₂ environment for 24 h. Cells were treated with 20 μg/ml of AC for 6, 9, 12, 15 h. The cells were harvested with ice-cold PBS and the cell pellets were incubated with cytosolic lysis buffer (10 mM HEPES, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, pH7.6) and centrifuged to get cytosolic fractions. The insoluble fractions were re-suspended with nuclear lysis buffer (20 mM HEPES, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM DTT) and centrifuged to isolate nuclear fractions. Whole cell lysates were prepared with RIPA buffer (25 mM Tris, 150 mM NaCl, 0.5% Triton X-100). The lysates were quantified with BCA protein assay kit (Pierce, Rockford, IL, USA) and loaded on SDS-PAGE after denaturation. The proteins were blotted to PVDF membrane and probed with the primary antibodies. The membranes were then incubated with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (Cell Signaling, Beverly, MA, USA) as the secondary antibody and visualized using the ECL chemiluminescence (GE healthcare, Piscataway, NJ, USA). All the primary antibodies were used in 1:1000 dilution and the secondary antibodies in 1:10000.