Anti phospho stat2

Approximately 30 mg of mouse liver was homogenized and centrifuged at 16,500 g for 20 minutes at 4°C. After adding sodium dodecyl sulfate (SDS) sample buffer, the supernatants were boiled for 10 minutes at 95°C. Protein concentrations in the samples were determined by the BCA assay (Thermo Fisher Scientific Inc.). Then, appropriate amounts of protein were loaded onto 8–12% SDS–polyacrylamide gels and electrotransferred onto a polyvinylidene fluoride membrane. The membrane was blocked with 5% skimmed milk or 1% bovine serum albumin for 1 hour at room temperature and then incubated overnight at 4°C with the primary antibodies anti-STAT1, anti-phospho-STAT1, anti-IRF3, anti-phospho-IRF3 (diluted 1:1000, Cell Signaling Technology), anti-STAT2, anti-phospho-STAT2 (diluted 1:1000, Abcam, Cambridge, UK) or β-actin (diluted 1:10,000, Sigma-Aldrich, St. Louis, MO, USA). After the membrane was washed three times for 5 minutes in TBST, it was incubated with the appropriate HRP-conjugated secondary antibody (diluted 1:5000 in TBST) for 1 hour at room temperature. Blotted protein bands were visualized by enhanced chemiluminescence (Thermo Fisher Scientific Inc.) and exposed to X-ray film.

Cells were lysed with RIPA buffer supplemented with a protease inhibitor cocktail (Sigma, Shanghai, China). The protein concentration was determined using a BCA protein assay kit (Bestbio, Shanghai, China). Aliquots of total cell lysates (40 μg protein) were mixed with loading buffer, boiled for 5 min, and subjected to 10% SDS-PAGE. Proteins were blotted onto nitrocellulose membranes. The membranes were blocked with 5% bovine serum albumin and then incubated at 4°C overnight with anti-phospho-STAT1 (Abcam, Cambridge, MA, USA), anti-STAT1 (Abcam), anti-phospho-STAT2 (Abcam), anti-STAT2 (Abcam), anti-phospho-STAT3 (SAB Signalway Antibody, College Park, MD), anti-STAT3 (SAB), anti-GAPDH (Abcam), anti-IFNAR1 (Proteintech, Chicago, IL, USA), and anti-ubiquitin (Santa Cruz Biotech) antibodies. Next, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotech) and developed using an enhanced chemiluminescence detection system (Amersham Bioscience, Piscataway, NJ, USA). The intensity of each signal was determined by a computer imaging analysis system (Quantity One, Bio-Rad, Hercules, CA, USA).

DMEM (25 mmol/L glucose), DMEM (5.5 mmol/L glucose), NuPAGE® Novex® Bis-Tris Gels, NuPAGE® LDS sample buffer (4×) and anti-STAT2 were acquired from Thermofisher Scientific. BSA, fibronectin, transferrin, sodium selenite, nicotinamide, Tris, NaCl, EDTA, Triton-X100, protease inhibitor, phosphatase inhibitor cocktails 2 and 3, IGEPAL, PVDF membrane, paraformaldehyde and protein G sepharose beads were purchased from Merck. Gamma-interferon activation site (GAS) and interferon stimulation response element (ISRE) reporter construct were from Qiagen. Mirus TransIT-2020 transfection reagent was obtained from Cambridge Bioscience (Cambridge, UK). Anti-STAT1 was obtained from Cell Signalling Technology. Anti-phospho STAT1 and anti-phospho STAT2 were acquired from Abcam. Anti-GAPDH was from Proteintech (Manchester, UK). Isotype control anti-mouse IgG was from Dako.