Anti phospho irf3

Polymyristate-13-acetate (PMA), Polyinosinic–polycytidylic acid (poly (I: C)), Herring testis (HT) DNA, and dimethyl sulfoxide (DMSO) were all purchased from Sigma-Aldrich (Jefferson City, United States). 2'3'-cGAMP were purchased from InvivoGen. Penicillin-streptomycin 100× sterile (CC004) was purchased from MacGene (Beijing, China). StarFect High-efficiency Transfection Reagents were purchased from GenStar. DMXAA (HY-10964). DiABZI STING agonist-1 trihydrochloride (HY-112921B) was purchased from Med Chem Express (State of New Jersey, US). Rabbit monoclonal anti-Phospho-IRF-3 (1:1000,86691) was purchased from Gene Tex (China). Rabbit monoclonal anti-Phospho-IRF-3 (1:1000,76439) was purchased from Abcam. TMEM173/STING Polyclonal antibody (1:2000,19851-1-AP), IRF3 Polyclonal antibody (1:2000,11312-1-AP), Alpha Tubulin Monoclonal antibody (1:1500, 66031-1-Ig) and Anti Lamin B (1:1500, 66095-1-Ig) antibodies were purchased from Proteintech (Chicago, United States).

IECs were lysed in RIPA buffer (150 mM NaCl, 0.5% sodium deoxycholate, 1% NP40, 0.1% SDS, 50 mM Tris-HCl [pH 8.0]) containing protease inhibitor and phosphatase inhibitor cocktail (Roche). Cell extract of 80 μg was separated by 10% SDS-PAGE and transferred to PVDF membranes, which were incubated with primary antibodies, then horseradish peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratory, West Grove, PA, USA) and visualized using an enhanced chemiluminescence system (ThermoFisher Scientific). Images were acquired using a digital image system (UVP BioSpectrum). The antibodies were anti-AiV VP1 [13 (link)], anti-phospho-IFN regulatory factor 3 (anti-phospho-IRF3, Abcam), anti-IRF3 (Santa Cruz Biotechnology, Houston, TX), anti-mitochondrial antiviral-signaling protein (anti-MAVS; Abcam, Cambridge, UK), and anti-β-actin (ThermoFisher Scientific) as the internal loading control.

Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, NP-40, 1 mM EDTA, and 1× protease inhibitor cocktail [Roche, Mannheim, Germany]) according to the manufacturer’s protocol. The samples were separated with sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane, and probed with the indicated antibodies. Where indicated, Western blot signals were quantified by densitometry as previously described (51 (link), 52 (link)). The following antibodies were used: anti-polyclonal-NP (produced in our lab), anti-phospho-IRF3 (Abcam, Cambridge, UK), anti-IRF3 (Proteintech, Wuhan, China), anti-STAT1 (Abcam), anti-phospho-STAT1 (Abcam), anti-β-actin (TranGens Biotech, Beijing, China), anti-p65 (Abclonal, Wuhan, China) and anti-phospho-p65 (Abclonal).