Anti stat1

The cells were stimulated with respective cytokines (IL-6 - 10 ng/ml, IFNγ - 2.5 ng/ml) for half an hour. Protein lysates were prepared in IP buffer (25 mM HEPES, 25 mM Tris-HCl, 150 mM NaCl, 10 mM EDTA, 0.1% Tween-20, 0.5% NP-40; with protease and phosphatse inhibitors). Protein A Sepharose CL4B beads (GE Healthcare) were blocked with 2% BSA and resuspended in IP buffer. Immunoprecipitation was done overnight (at 4°C) with 1 mg of protein and 10 μg of STAT3 antibody (sc-482, Santa Cruz) in 10 mM HEPES (pH7.5) and 1 mM EDTA (5 times the volume of IP buffer). The beads were washed thrice with the HEPES-EDTA buffer. The beads were boiled with loading buffer to extract the bound immunoprecipitated protein and the supernatant was analysed by Western blotting using anti-STAT3 (BD #610189) and anti-STAT1 (BD #610115) antibodies.

Infected tissues were lysed with RIPA buffer at 2 dpi. 15μg of lysed proteins were loaded on a 10% SDS-PAGE gel and then transferred onto PVDF membrane (Bio-Rad). The membranes were first blocked with 5% skim milk (AppliChem) in TTBS (10 mM Tris HCl, pH 7.5, 500 mM NaCl, 0.05% Tween 20) at RT for 30min and then incubated with primary Ab overnight at 4°C. The membranes were then washed thrice with TTBS and incubated with secondary antibodies conjugated to HRP at RT for 1h. The membranes were washed and later developed with Western Bright Sirius ECL system (Advansta Inc.) for 2 min. Immunoblot images were acquired using Fujifilm LAS 4000 luminescence imager. Primary antibodies used in Western blot include anti-Stat1 (612233, BD), -MDA5 (ENZ-ABS299-0100, Enzo Life Sciences), -MX1 (ab95926, Abcam), -IFIT2 (ab113112, Abcam) and -actin (MAB1501, Merck Millipore). All primary antibodies were diluted 1:1000 except MX1 diluted in 1:2000. Goat anti-rabbit (7074, Cell Signaling Technology) and horse anti-mouse (7076, Cell Signaling Technology) secondary antibodies conjugated to HRP were diluted in 1:5000 and used for chemiluminescence development.

The following phospho-specific monoclonal antibodies were used in 3 different panels during the flow cytometry protocol described previously (17 (link)): Alexa Fluor®647 conjugated anti-STAT4 (pY693, clone 38/p-STAT4, panel 1), anti-STAT 1 (pS727, clone K51-856, panel 2), and anti-STAT3 (pS727, clone 49/p-STAT3, panel 3); PerCP-Cy^TM 5.5 conjugated anti-ERK1/2 (pT202/pY204, clone 20A, panel 1), anti-STAT1 (pY701, clone 4a, panel 2), and anti-STAT3 (pY705, clone 4/P-STAT3, panel 3); and PE-Cy^TM7 conjugated anti-p38 MAPK (pT180/pY182, clone 36/p38, panel 2), and anti NF-κB p65 (pS529, clone K10-895.12.50, panel 1), anti-STAT5 (pY694, clone 47/STAT5(pY694), panel 3) (all from BD Biosciences, San Jose, CA, USA). Cell surface markers incorporated in the assays were BV786 conjugated anti-CD3 (clone SK7, BD Horizon^TM), Alexa Fluor® 488 conjugated anti-CD20 (clone H1 (FB1), BD Biosciences) and PE conjugated anti-CD56 (clone N901, Beckmann Coulter, CA, USA).

Monoclonal antibodies (Abs), specific for cluster of differentiation (CD)1a, CD14, CD38, CD86, CD83, HLA-DR, CD40, IgG1, and IgG2a (BD Bioscience, San Diego, CA, USA), were directly conjugated to fluorescein isothiocyanate (FITC) or phycoerythrin (PE). To exclude dead cells from the analysis, Fixable Viability Dye eFluor®780 (FvDye) (eBioscience, San Diego, CA, USA) was used. For immunoblotting analysis, rabbit anti-STING (Cell Signaling, Danvers, MA, USA # 2775), anti-IRF3 (Santa Cruz, Santa Cruz, TX, USA # sc-9082), anti-IRF7 (Santa Cruz, # sc-9083), anti-STAT1 (BD Bioscience, # 610186), anti-phospho STAT1 (Cell Signaling Technology, Leiden, The Netherlands, # 7649), anti-STAT2 (BD Transduction Laboratories, # 610188), anti-phospho STAT2 (R&D Systems, Minneapolis, MN, USA, MAB2890), mouse anti-actin (Sigma-Aldrich, St. Louis, MO, USA #A0483), and horseradish peroxidase-conjugated secondary antibody anti mouse (Santa Cruz, # sc-2005) and anti rabbit (Santa Cruz, # sc-2004) were used. For phagocytosis and phagosomal acidification experiments, cytochalasin D 5 μM (Sigma-Aldrich, # C8723) and chloroquine 2 μM (Sigma-Aldrich, # C6628) were used.