To correct for sample-to-sample variations in qRT-PCR efficiency and errors in sample quantitation, the level of GAPDH transcript was measured to normalize specific RNA levels. External standards were used to establish standard PCR curves for quantifying copies of transcripts that required

an absolute, comparative quantitation. Fold-changes in expression were determined by dividing the normalized quantity of the gene of interest from IFNα-treated or IFNγ-treated cells by the normalized quantity of the gene of interest from untreated cells. Total levels of STAT1, STAT2, P-STAT1, and P-STAT2 molecules were measured by immunoblot in protein extracts from IFN-treated and untreated cells. Antibodies specific for STAT1 (C-terminus), Raf inhibitor P-STAT1 (pY701), STAT2, P-STAT2 (pY690), were purchased from BD Biosciences, Venetoclax while the anti-mouse IgG (Fc specific)-peroxidase secondary antibody and the monoclonal anti-alpha-tubulin were from Sigma-Aldrich. Lysates were prepared from cells plated at 5 × 105 cells /well in 6-well plates with 2 ml of medium. Adherent cells were removed by brief treatment with trypsin and EDTA (Sigma-Aldrich) and

then combined with non-adherent cells from the same culture and washed in cold PBS prior to being resuspended in 100 μl of RIPA buffer (10 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1% NP40). Protease inhibitor cocktail tablets from Roche were added at 1× concentration immediately prior to Fenbendazole sample preparation. After 15 min of incubation at 4 °C with agitation, samples were centrifuged for 1 h at 4 °C and 12,500 rpm, and the recovered supernatant was divided into aliquots and stored at −80 °C until it was subjected to polyacrylamide gel electrophoresis. Protein concentrations were determined using a Bio-Rad protein assay (Bio-Rad Inc.) with bovine serum albumin standards, following the manufacturer’s

recommendations. Equal amounts of solubilized proteins (30 μg) were diluted in Laemmli sample buffer and subjected to electrophoresis on 12.5% acrylamide/bis gels. Proteins were then transferred onto PVDF membranes (Immobilon-P from Millipore) using an electroblotting system from Biometra. Membranes were prepared for immunoblotting by washing in TTBS (10 mM Tris–glycine, pH 8.0, 0.15 M NaCl, with 0.05% (w/v) Tween-20). Membranes were then blocked in TTBS plus 5% (w/v) non-fat dry milk for 1 h, followed by three 5 min washes in TTBS. Membranes were probed for specific proteins by 1 h incubations with the specific antibodies at the dilution suggested by the manufacturers. The membranes were then washed three times in TTBS and developed with the recommended dilution of the secondary antibody. After 1 h, the membranes were washed in TTBS, and the proteins on the nitrocellulose membrane were detected using the ECL Plus detection system (Healthcare/Amersham Biosciences), according to the manufacturer’s protocol.