Supersignal west pico chemiluminescent kit

Samples from skeletal muscle (25 µg), run on an SDS-PAGE gel, were transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA) through semi-dry transference, and protein content confirmed using Ponceau S staining. After three cycles of TTBS (Tris 100 mM pH 7.5; 0.9% NaCl, and 0.1% Tween-20) washing, membranes were blocked with 5% non-fat dry milk for 1 h at room temperature. After washing, membranes were incubated with primary antibodies for SOD1, SOD2, CAT, IL-1β, IL-10, TNF-α, heat-shock protein 70 (HSP70), and β-actin for 2 h at room temperature in a 1:1000 dilution range. Secondary antibodies (anti-rabbit/mouse/goat peroxidase-linked—Cell Signaling Technology, Beverly, MA, USA) were incubated for 1 h at room temperature in a 1:2000 dilution range. Detection of immunoreactivity was performed through chemiluminescence using a Supersignal West Pico Chemiluminescent kit (Thermo Scientific, Rockford, IL, USA). Densitometry analysis was conducted with ImageJ software (version 1.50i, National Institutes of Health, Bethesda, MD, USA), and the results were expressed as ratio of protein:β-actin.

Cells were treated with IL-6/sIL-6R for 0, 1, 6, 12, 24, and 48 hours. For another experiment, RASFs were cultured with IL-6/sIL-6R plus T-614 or MTX for 72 hours, using 25 μM PD98059 (CST, USA) before IL-6/sIL-6R was added as control. Proteins were extracted in lysis buffer (30 mM Tris [pH 7.5], 150 mM sodium chloride, 1 mM PMSF, 1 mM sodium orthovanadate, 1% nonidet P-40, 10% glycerol, and phosphatase and protease inhibitors). The proteins were then separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes. The membranes were probed with RANKL (EPR4999, Abcam, Cambridge, MA, USA), p-ERK1/2 (D13.14.4E, Cell Signaling, MA, USA), ERK1/2 (137F5, Cell Signaling, MA, USA) or GAPDH (D16H11, Cell Signaling, MA, USA), or β-actin (13E5, Cell Signaling, MA, USA) overnight at 4°C and then incubated with an HRP-coupled secondary Ab (Cell Signaling, MA, USA). Proteins were detected with the SuperSignal West Pico Chemiluminescent Kit (Thermo Scientific, Rockford, IL, USA). Densitometry values were analyzed and quantified with ImageLab (Bio-Rad).

The cells (1 × 10⁶ treated for 6 h) post treatments were washed with HBSS followed by homogenization in 2 mL of RIPA buffer/10% of Protease Inhibitor (Sigma-Aldrich, AUS). After subjecting to centrifugation at 12,000 rpm for 20 min at 4 °C, the supernatant was collected. Thirty micrograms of protein from each sample was denatured in Laemmli loading buffer (Bio-Rad Laboratories, AUS) and separated on precast 12% SDS-PAGE gels (Bio-Rad Laboratories, AUS), which was followed by overnight transfer onto Polyvinylidene difluoride (PVDF) membranes (Millipore, NSW, AUS) at 30 mV at 4 °C. By using 5% non-fat milk, the blot was blocked before incubating with respective antibodies: anti-GADPH, Survivin, Grp78 (#14C10, 1:3000, Novus Biologicals, AUS), poly(ADP-ribose)polymerase (PARP), and cleaved PARP (SIGMA, VIC, AUS) overnight at 4 °C in blocking buffer. The blot was washed in PBST and incubated with appropriate species monoclonal horseradish peroxidase-conjugated anti-IgG secondary antibodies (1:5000) for 1 h at 20 °C. The bands were visualized by using the Supersignal West Pico chemi-luminescent kit (Thermo Scientific, VIC, AUS). It was digitized and the band intensities were determined by using a Fuji LAS-3000 Imager (Fuji Life Sciences, JPN). The samples from all groups were included in each individual blot to ensure accurate quantification across multiple blots.