Anti phospho stat1

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.

The antibodies used in this study were as follows: anti-GST (Santacuze, #SC-138), anti-V5 (Invitrogen, #46–0705), or anti-IRF3 (Abcam, #ab25950), anti-phospho-IRF3 (Ser 396) (Cell signaling, #4947), anti-NF-κB p65 (Cell signaling, #4764), anti-phospho-NF-κB p65 (Ser536) (Cell signaling, #3031), anti-STAT1 (Cell signaling, #9175), anti-phospho-STAT1 (Cell signaling, #9167), anti-phospho-p38 (Cell signaling #9216), phospho-TBK1 (Cell signaling #5483), anti-NLRX1 (Proteintech, #17215-1-AP) and anti-His (Santacuze, #SC-1803) antibodies. The anti-FAF1 monoclonal antibody was provided by Dr. Eun-hee Kim (Department of Biology, Chungnam National University, Korea). The anti-interferon-α/β receptor (IFNAR) (25 μg/ml; Leinco Technologies) was pre-incubated in RAW264.7 cells and MEFs for 1 hr before VSV-GFP infection to block IFNAR.

Total proteins, cytosolic and nuclear extracts were prepared as previously reported [24] (link), and subjected to SDS-PAGE. Western blotting filters were developed using the ECL-plus detection system (Amersham, Dubendorf, Switzerland) or the SuperSignal West Femto kit (Pierce, Rockford, IL). The Abs employed for the study were as follows: anti-phospho-STAT3 (Serine 727, Ser727), anti-STAT3 (C-20), anti-phospho-STAT1 (Tyrosine 701, Tyr701), anti-STAT1 (E-23), anti-phospho-ERK1/2 (E-4), anti-ERK1/2 (C16), anti-phospho-EGFR (Tyr1173), anti-EGFR (1005), anti-Akt (H-136), anti-PCNA (PC10), anti-cyclin D1 (DCS-6), anti-β-actin (C-11), all provided by Santa Cruz Biotechnology (Santa Cruz, CA). anti-phospho-STAT3 (Tyr705), anti-acetyl-STAT3 (lysine 685, Lys685), anti-phospho-STAT1 (Ser727), anti-phospho-Akt (Ser473), and anti-phospho-Retinoblastoma (RB) (Ser795) were from Cell Signaling Technology (Denvers, MA). Filters were properly developed with anti-mouse or anti-rabbit Ig Abs conjugated to HRP.

Immunoblotting was performed according to standard procedures (see, e.g., see reference 53 (link)) using the following antibodies: anti-phospho-STAT1 (Cell Signaling Technology), anti-STAT1 (Santa Cruz), anti-STAT2 (Santa Cruz), anti-actin (Sigma), and anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase; Santa Cruz). Proteins were visualized using peroxidase-coupled secondary antibodies and an enhanced chemiluminescence system (Cell Signaling Technology).

Lysates (40 μg) of freshly isolated or cultured cells were analyzed with anti-p21 (Santa Cruz) anti-phospho ERK antibodies (Cell Signaling); anti-ERK (Cell Signaling), anti-CDK2 (Santa Cruz) and anti-β-actin (Sigma) served as loading controls. Macrophage whole cell lysates (20 μg) were probed with anti-phospho-STAT1 (Cell Signalling) and anti-iNOS (Abcam).

In order to make sure the loading amounts of the protein were comparable, the protein concentration was quantified by Bradford assay [25 (link)]. Thirty micrograms of protein was separated by SDS polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride (PVDF, Thermo Scientific) membranes. After blocking with 5% gelatin in TBST, the membranes were incubated with the indicated primary antibodies at 4 °C overnight. The membranes were washed three times, incubated with secondary antibodies (1:8000) conjugated with horseradish peroxidase (HRP) for 1 h at room temperature, and visualized with the ECL system (Biorad, Hercules, CA, USA) according to manufacturer’s instructions. Blots were probed with HRP-conjugated secondary antibodies. The following antibodies were used: anti-IFITM1, 2, 3, (catalog no.13126, 13530, 59212, Cell Signaling Technology, Danvers, MA, USA), anti-BAF180 (catalog no. A301-591A, Bethyl), anti-STAT1 (catalog no. 9172, Cell Signaling Technology), and anti-phospho-STAT1 (catalog no. 9167, Cell Signaling Technology), and β-actin (catalog no. 60008-1-Ig, Proteintech Group). The gray density of the western blots was measured by using ImageJ software (National Institutes of Health, Bethesda, MD, USA).