Phosphorylated irf3

Cells, after being washed three times with PBS, were lysed with lysis buffer (Beyotime, Shanghai, China) for 20 min on ice and collected for the bicinchoninic acid method (Beyotime, Shanghai, China) to quantify the lysates. Protein lysate samples were subjected to SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Merck Millipore, Billerica, MA, USA). After being blocked by 5% fat-free milk (Sangon Biotech, Shanghai, China) in TBST (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.05% Tween 20) for 2 h at room temperature, membranes were incubated with indicated protein antibodies diluted in a ratio of 1:1,000 at 4°C overnight. The antibodies used in the study include anti-PRRSV N (MEDIAN, Republic of Korea) antibody, anti-GAPDH, RIG-I, MDA5, mitochondrial antiviral-signaling protein (MAVS), p65, phosphorylated p65, IRF3, phosphorylated IRF3, phosphorylated IκBα (Cell Signaling Technology, Danvers, MA, USA) antibodies, and anti-nsp2 antibody was provided by Prof. Yanhua Li from Yangzhou University. The membranes were incubated with corresponding secondary antibodies diluted in a ratio of 1:8,000 for 1 h and then washed with TBST three times. The protein signals were visualized using an enhanced chemiluminescence reagent (NCM Biotech, Suzhou, China) on a Tanon 5200 system (Tanon, Shanghai, China).

Antibodies used were as follows: TRF2 (Karlseder laboratory), ZBP1 (Novus Biologicals, NBP1-76854 and Cell Signaling Technology, 60968), LC3 (Cell Signaling Technology, 2775 and 3868), STING (Cell Signaling Technology, 13647), CGAS (Cell Signaling Technology, D1D3G), γH2AX (Millipore, 05-636-I), Flag (Sigma-Aldrich, F1804), GAPDH (Abnova, PAB17013), TBK1 (Cell Signaling Technology, 3504S), phosphorylated TBK1 (Cell Signaling Technology, 5483S), STAT1 (Cell Signaling Technology, 9172S), phosphorylated STAT1 (Cell Signaling Technology, 9167L), IRF3 (Cell Signaling Technology, 11904S), phosphorylated IRF3 (Cell Signaling Technology, D4947S), MAVS (Cell Signaling Technology, 24930S), TOMM20 (Abcam, ab56783), TFAM (Abcam, ab119684), dsDNA (Abcam, ab27156), dsRNA (Millipore, MABE1134), phosphorylated ATR (Abcam, ab223258), phosphorylated ATM (Cell Signaling Technology, 5883), the apoptosis antibody sampler kit (Cell Signaling Technology, 9915), the necroptosis antibody sampler kit (Cell Signaling Technology, 98110) and the pyroptosis antibody sampler kit (Cell Signaling Technology, 43811).

DMEM medium, RPMI-1640, penicillin-streptomycin and Lipofectamine 2000 were acquired from Invitrogen Life Technologies. Fetal bovine serum (FBS) was purchased from HyClone. Polyinosine-polycytidylic acid [Poly (I:C)] was obtained from Invivogen. Lipopolysaccharide (LPS) and M2 beads were purchased from Sigma. Recombinant mouse IFN-β was obtained from Sino Biological. TRIzol reagent and PrimeScript RT Master Mix were purchased from Takara. SYBR Green PCR Master Mix was purchased from Yeasen. Dual-Luciferase Reporter assay reagent was purchased from Promega. Antibodies specific to TBK1, phosphorylated TBK1, IRF3, phosphorylated IRF3, ERK, phosphorylated ERK, P38, phosphorylated P38, JNK, phosphorylated JNK, AKT, and phosphorylated AKT were obtained from Cell Signaling Technology. Polyclonal anti-GAPDH, anti-HA, anti-Flag antibody were obtained from Biogot technology. HA beads were obtained from Abmart. INT-777 (a novel potent and selective TGR5 agonist) was purchased from MedChem Express.