Supersignals west pico chemiluminescent substrate

The other half of the spleen of each mouse was lysed in 0.5 mL of a lysis buffer (Sigma). The extracts were cleared by centrifugation at 10,000 g at 4°C for 15 minutes and then diluted with lysis buffer to achieve approximately 2 mg/mL protein concentration. Protein samples were separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes (Amersham Pharmacia Biotech, Uppsala, Sweden). The membranes were incubated with primary antibodies, including anti-T-bet/Tbx21 antibody (1:1000), (Abcam, Cambridge, MA, USA), anti-mouse GATA-3, RORγ, Jak1, Stat-1 and Stat-3 rabbit monoclonal antibodies (CST, Boston, MA, USA), and were then incubated with a horseradish peroxidase-conjugated secondary antibody (CST). All immunoreactive proteins were visualized with SuperSignals west Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA). Densitometry plots showing the protein expression levels were normalized to GAPDH and expressed in terms of the fold change relative to the levels in the normal group.

The other half of the spleens of CIA mouse was homogenized in 1 ml of a lysis buffer (Sigma, CA, USA). The extracts were cleared by spinning at 10,000 g at 4 °C for 15 min, and then diluted with the lysis buffer to achieve approximately 2 mg/ml protein concentration. Protein samples were separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membranes (Amersham Pharmacia Biotech, Uppsala, Sweden). The membranes were incubated with primary antibodies, including anti- NF-κB kinase inhibitor (IKK)α/β, anti-NF-κB p50, anti- glycoprotein (gp)-130, anti-JAK1 and anti-STAT3 rabbit anti-mouse monoclonal antibodies (CST, Boston, MA, USA), and were then incubated with horseradish peroxidase-conjugated secondary antibody. All immunoreactive proteins were visualized with SuperSignals west Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA). Densitometry plots showing the expression of proteins were normalized to glyceraldehyde 3 - phosphate dehydrogenase (GAPDH) and expressed as fold relative to the levels in mice of the normal group.

The protein expression levels of caspase-3, cleaved caspase-3, caspase-8, cleaved caspase-8, Fas, Stat-1, and Stat-3 and the phosphorylation of Stat-1, and Stat-3 were analyzed by Western blotting. After 12 h or 24 h of treatment, BMCs were washed with PBS three times and lysed in RIPA buffer with protease inhibitor cocktail. The protein samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then were electrotransferred to nitrocellulose membranes. The membranes were incubated with 1:1000 dilutions of anti-mouse caspase-3, caspase-8, cleaved caspase-3, cleaved caspase-8, Fas, Stat-1, Stat-3, phospho-Stat-1, and phospho-Stat-3 rabbit monoclonal antibodies (CST, Boston, MA, USA) at 4°C overnight. Then, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (CST) for 1 h at room temperature. The immunoreactive proteins were detected with Super Signals west Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA). Densitometry plots showing protein expression were normalized to β-actin.