Anti p stat1 y701

Cells were lysed using Pierce RIPA buffer supplemented with 1% PMSF and phosphatase inhibitors (KeyGEN BioTECH). Protein concentration was measured using the BCA Protein Assay (Thermo Scientific). Equal amounts of protein were separated on 10% SDS-PAGE and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). After blocking, the membrane was incubated with a primary antibody at 4 °C overnight and subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at room temperature. The following primary antibodies were used: rabbit monoclonal anti-STAT1, anti-pSTAT1-Y701, anti-pSTAT1-S727, mouse monoclonal anti-Smad2, rabbit polyclonal anti-pSmad2, anti-β-actin (Cell Signaling Technology), rabbit polyclonal anti-HA and anti-c-Myc (Santa Cruz Biotechnology, Inc., CA, USA). Signals were detected using Immobilon™ Western Chemiluminescent HRP Substrate (Millipore) and quantified using Tanon-4500 Gel Imaging System with GIS ID Analysis Software v4.1.5 (Tanon Science and Technology Co., Ltd., Shanghai, China).

The following primary antibodies were used for Western blotting: rabbit anti-pPKD1 S744/S748 (pPKD1/2 activation loop), rabbit anti-pPKD1 S916, rabbit anti-PKD1, rabbit anti-PKD2, anti-pSTAT1 Y701, rabbit anti-STAT1 (Cell Signaling), rabbit anti-lamin B1 (LB1) (Proteintech), and rabbit anti-pPKD2 S876 (Millipore). The rabbit antibody to HRV 2C was generated and used as previously described (9 (link)). Secondary antibodies conjugated to horseradish peroxidase (HRP) were obtained from Jackson ImmunoResearch and were revealed by using the ECL reagent (Geneflow). PDBu) (Sigma) was dissolved in DMSO and used at a final concentration of 200 nM in all the experiments as a positive control for PKD phosphorylation. For the interferon receptor (IFNAR)-blocking antibody studies, either a mouse anti-IFNAR2 antibody (Stratech) or isotype control mouse IgG2a (Abcam) was used at a 5-μg/ml final concentration. Recombinant human IFN-β1α (IFN-β; R&D) was used at a final concentration of 30 U/ml. The primary antibodies used for confocal microscopy were mouse anti-Myc (Merck Millipore) and rabbit anti-GM130 (BD Pharmingen) in combination with a donkey anti-rabbit secondary antibody coupled to Alexa 546 and a donkey anti-mouse secondary antibody coupled to Alexa 488, respectively (Jackson ImmunoResearch).

Total cellular proteins were isolated using standard methods [20] (link). In brief, cell monolayers were washed with PBS and treated with lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 50 mM NaF, 10 mM β-glycerophosphate, 0.1 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM sodium orthovanadate) supplemented with a protease inhibitor cocktail (GE Healthcare). The lysates were then clarified by centrifugation at 13,000 g for 10 min at 4°C and the protein concentrations of the whole cell lysates were measured by the Bradford protein assay (Bio-Rad laboratories, Inc.). Lysates of the same number of cells (around 40 µg protein) were subjected to electrophoresis on SDS-PAGE gels followed by Western blot according to standard techniques, using the following monoclonal antibodies: anti-Nitric Oxide Synthase, Inducible (Sigma-Aldrich), anti-phospho(p)-NF-κB p65 (Ser536), anti-IκB-α, anti-STAT-1, anti-p-STAT-1 (Y701) and anti-β-actin (Cell Signaling Technology Inc.). Peroxidase-labeled anti-Rabbit IgG (KPL) was used as the secondary antibody. Immunoreactive bands were visualized using a Luminol chemiluminescent HRP substrate (Millipore) and were analyzed in a Storm System 860 (GE Healthcare). Densitometry analyses were performed using ImageJ 1.44p (National Institutes of Health, Bethesda, MD).