Anti irf3

HEK293T cells (ATCC CRL11268), 293-Dual hSTING-A162 cells (InvivoGen; 293d-a162), PK-15 cells (ATCC CCL-33), A549 cells (ATCC CCL-185), Vero cells (ATCC CCL-81), and MA104 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Cytiva) and PAMs (ATCC CRL2843) in RPMI medium (Cytiva). Cells were supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% antibiotic/antimycotic (Gibco) and incubated in a humidified 5% CO₂ incubator at 37°C. Antibodies used for the immunoblot and immunoprecipitation analysis are as follows: anti-Flag (Cell Signaling; 8146), anti-GST (Santa Cruz; sc-138), anti-Cy5 (Cell Signaling; sc-166896), anti-IRF3 (Abcam; ab25950), anti-phospho-IRF3 (Ser396) (Cell Signaling; 4947), anti-p65 (Cell Signaling; 4764S), anti-phospho-p65 (Cell Signaling; 3031S), anti-TBK1 (Cell Signaling; 3504S), anti-phospho-TBK1 (Cell Signaling; 5483S), anti-phospho-IκBα (Cell Signaling; 2859S), anti-IκBα (Cell Signaling; 9242S), anti-STING (Cell Signaling; 3337S), and anti-β-actin (Santa Cruz; SC 47778).

Protein samples were run on pre-cast NuPAGE™ Bis-Tris gels (Invitrogen) with 1 x MOPS buffer (Invitrogen) and transferred on nitrocellulose membranes, using iBlot^®2 Gel Transfer Device (Invitrogen). Membranes were washed in Tris Buffered Saline with 0.1% Tween-X100 (TBS-T) and blocked with TBS-T containing 5% bovine serum albumin (BSA, Sigma-Aldrich). Membranes were incubated with primary antibodies at 4°C overnight. The following primary antibodies were used: GAPDH (1:5000; ab8245; Abcam), anti-β-Actin (1:5000; cat#6276; Abcam) anti-IRF3 (1:1000; D83B9; cat#4302S; Abcam), anti-TBK1 (1:1000; cat#3504; Cell Signaling Technology), anti- IL1B (1:1000; cat#12242; Cell Signaling Technology) and SQSTM1 (1:1000; cat#PM045; MBL). Membranes were washed in TBS-T and incubated with secondary antibodies (HRP-conjugated, DAKO) for 1 hour at room temperature in TBS-T containing 1% milk or BSA. The blots were developed with SuperSignal West Femto Substrate (Thermo Scientific) and captured with LI-COR Odyssey system (LI-COR Biosciences, Lincoln, NE, USA).

RAW264.7 (ATCC TIB-71), HEK293T (ATCC-11268), HeLa (ATCC CCL-2), PK-15 (ATCC CCL-33), LFBK (RRID:CVCL_RX26), cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (HyClone) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% antibiotic/antimycotic (Gibco). Cells were maintained in a humidified 5% CO2 incubator at 37°C. Antibodies used for the immunoblot and immunoprecipitation analysis are as follows, anti-Flag (Cell Signaling, 8146), anti-Strep (Qiagen, 34850), anti-GST (Santa Cruz, sc-138), anti-IRF3 (Abcam, ab25950), anti-phospho IRF3 (Ser396) (Cell Signaling, 4947), anti-p65 (Cell Signaling, 4764S), anti-phospho p65 (Cell Signaling, 3031S), anti-TBK1 (Cell Signaling, 3504S), anti-phospho-TBK1 (Cell Signaling, 5483S), anti-β-actin (Santa Cruz,SC 47778), RIG-I (D14G6; 3743), MDA-5 (D74E4; 5321), Anti-FMDV 2B (homemade), anti-Caspase8 (Cell Signaling, 9746S), anti-Caspase3 (Cell Signaling, 9662S) and anti-β-actin (Santa Cruz,SC 47778)

RAW264.7 cells were treated with DMEM alone (negative control), DMEM with 100 ng/ml LPS (positive control), or DMEM with 1.0 μg/ml CP, and cells were harvested at 0, 3, 6, 12, and 24 hpt. Cell pellets were washed with PBS and whole cell lysates were subjected to SDS-PAGE followed by standard immunoblotting with indicated antibodies: anti-IRF3 (Abcam, #ab25950), anti-phospho-IRF3 (Ser396) (Cell Signaling, #4947), anti-p65 (Cell Signaling, #4764S), anti-phospho-p65 (Cell Signaling, #3031S), anti-STAT1 (Cell Signaling, #9175), anti-phospho-STAT1 (Cell Signaling, #9167), anti-TBK1 (Cell Signaling, #3504S), or anti-phospho-TBK1 (Cell Signaling, #5483S), anti-p38 (Cell Signaling, #9212), anti-phospho-p38 (Cell Signaling #4631S), anti-ERK (Cell Signaling, #9102), anti-phospho-ERK (Cell Signaling, #9102S), or anti-β-actin (Santa Cruz SC 47778).

Total proteins were extracted using cell lysis buffer (BioFeng, Changsha, Hunan, China). After the protein concentrations were determined by BCA kits, the protein samples were extracted and separated by 10% SDS-PAGE gel and then transferred to a PVDF membrane (Millipore, USA). The membrane was then blocked with 5% skimmed milk and incubated overnight, using the following main detection antibodies at 4°C: anti-GAPDH (1 : 1,000; Abcam), anti-Histon-H3 (1 : 1,000; Abcam), anti-Hsp60 (1 : 1,000; Abcam), anti-cGAS (1 : 1,000; Abcam), anti-STING (1 : 1,000; Abcam), anti-pSTING (1 : 1,000; Abcam), anti-TBK1 (1 : 1,000; Abcam), anti-pTBK1 (1 : 1,000; Abcam), anti-IRF3 (1 : 1,000; Abcam), anti-pIRF3 (1 : 1,000; Abcam), anti-PD-L1 (1 : 1,000; Abcam), anti-CD9 (1 : 1,000; Abcam), anti-TSG101 (1 : 1,000; Abcam), anti-Alix (1 : 1,000; Abcam), anti-Hsp90 (1 : 1,000; Abcam), or anti-β-actin (1 : 5,000; Proteintech). We washed 3 times with TBS-T, and the membranes were cultured with the secondary antibody at 24°C for 1 hr. The western blots were pictured using an ECL Reagent (Pierce, USA), and the density was verified using ImageJ software (NIH, USA).

Anti‐TBK1 (1:1,000; Thermo Fisher Scientific, Rockford, IL, PA5‐17478), anti‐IRF3 (1:1,000; Abcam, MA, #ab25950), anti‐pIRF3 (Ser396) (1:500 Cell Signaling, MA, 4D4G, #4947), anti‐OPTN(1:1,000, Proteintech, 108371‐AP), and anti‐β‐actin (1:10,000; Sigma, #A5060) antibodies were used. The following secondary HRP‐conjugated antibodies were used for immunoblotting: goat anti‐mouse IgG (1:1,000; Santa Cruz, CA, #sc‐2005) and goat anti‐rabbit IgG (1:1,000; Santa Cruz, #sc‐2004).

HEK293T cells were transfected with appropriate plasmids and lysed by co-immunoprecipitation lysis buffer (10% glycerol, 0.5% NP-40, 150 mM NaCl, 0.1 mM EDTA) with protease inhibitor cocktail (Roche). Cell lysates were incubated with anti-FLAG antibody (Sigma, F3165) and protein A/G (Santa Cruz Biotechnology, sc-2003). The beads were washed by PBSN (PBS containing 0.1% NP-40) three times and subjected to SDS-Page. For subcellular fractionation, nuclear and cytoplasmic extracts were isolated with a nuclear-cytoplasmic extraction kit (Applygen, P1200) following the manufacturer’s protocol.
Antibodies used in this study were as follows: anti-RIG-I (Santa Cruz Biotechnology, sc-376845, 1:1000), anti-p-IRF3 (Cell Signaling Technology, #4947, 1:1000), anti-MDA5 (Abclonal, A2419, 1:500), anti-MAVS (abcam, ab189109,1:2000), anti-IRF3 (abcam, ab68481, 1:2000), anti-DDX3X (Santa Cruz Biotechnology, sc-365768, 1:500), anti-DHX9 (Santa Cruz Biotechnology, sc-137232, 1:500), anti-GAPDH (RayAntibody, RM2002, 1:5000), anti-α-tubulin (RayAntibody, RM2007, 1:5000), anti-HDAC1(Santa Cruz Biotechnology, sc-8410, 1:1000), anti-FLAG (Sigma, F3165, 1:5000), anti-GFP (RayAntibody, RM1008, 1:5000), anti-GFP (RayAntibody, RM1008), KPL peroxidase-labeled antibody to mouse IgG (H + L) (Seracare, 5220-0341, 1:5000) and KPL peroxidase-labeled antibody to rabbit IgG (H + L) (Seracare, 5220-0336, 1:5000).