Dual-specific luciferase assay kit (Promega), SYBR (Bio-Rad), polybrene (Millipore), poly(I:C) (InvivoGen), RNase A (Thermo Fisher), DNase I (Sigma), mouse antibodies against HA-tag (BioLegend), FLAG-tag (Sigma), Myc-tag (Cell Signaling Technology) and β-actin (Sigma), anti-phospho-IRF3 (Ser396) (Cell Signaling Technology), anti-IRF3 antibody (FL-425) (Santa Cruz Biotechnology), ML-60218 (MCE) were purchased from the indicated manufacturers. Rabbit anti-porcine RIG-I antibody was raised against recombinant porcine C-terminal RIG-I fragment. Mouse monoclonal anti-p30 antibody was raised against a recombinant ASFV CN/GS/2018 p30 protein. SeV, HSV-1 and EMCV were previously described [40 (link),41 (link)]. ASFV CN/GS/2018 strain was propagated on PAMs as previously described [17 (link)] by the African Swine Fever Regional Laboratory of the Lanzhou Veterinary Research Institute. HEK293T, THP-1 and PK-15 cells were purchased from ATCC. PAMs were prepared by bronchoalveolar lavage as previously described [42 (link)]. PAMs were cultured in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Thermo Fisher Scientific) at 37°C with 5% CO2.
Anti phospho irf3 ser396
Manufactured by Cell Signaling Technology
Sourced in China
Anti-phospho-IRF3 (Ser396) is a primary antibody that recognizes the phosphorylated form of the transcription factor IRF3 at serine 396. This antibody is designed for use in Western blotting to detect the activation of the IRF3 signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Anti irf3, by Cell Signaling Technology (5 mentions)
Anti p65, by Cell Signaling Technology (4 mentions)
Anti phospho p65, by Cell Signaling Technology (4 mentions)
Anti β actin, by Santa Cruz Biotechnology (4 mentions)
Anti phospho tbk1, by Cell Signaling Technology (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Anti tbk1, by Cell Signaling Technology (3 mentions)
Anti irf3, by Abcam (3 mentions)
Hek293t, by American Type Culture Collection (2 mentions)
Poly 1 c, by InvivoGen (2 mentions)
Anti tubulin, by Merck Group (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Anti flag, by Cell Signaling Technology (2 mentions)
Anti gst, by Santa Cruz Biotechnology (2 mentions)
Anti sting, by Proteintech (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Anti stat1, by Cell Signaling Technology (2 mentions)
Dnase 1, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
12 protocols using anti phospho irf3 ser396
1
Porcine RIG-I Activation Assay
2
Characterizing SARS-CoV-2 Immune Responses
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IFN-β and IFN-λ1 were purchased from BioLegend. SeV was purchased from Charles River, and Poly(I:C) was purchased from Invivogen. Ruxolitinib was purchased from Selleck Chemicals. The following antibodies were used for Western blotting or immunofluorescence: anti-RIG-I (Cell Signaling Technology, 3743S), anti-MDA-5 (Cell Signaling Technology, 5321S), anti-MAVS (Cell Signaling Technology, 3993S), anti-IRF3 (Cell Signaling Technology, 11904S), anti-Phospho-IRF3 (Ser396) (Cell Signaling Technology, 4947S), STAT1 (Cell Signaling Technology, 14994), p-STAT1 (Cell Signaling Technology, 9167), anti-SARS-CoV-2 nucleocapsid (GeneTex, GTX135357), anti-SARS-CoV-2 ORF6 (Novus Biologicals, NBP3-05707), anti-SARS-CoV-2 Spike (Absolute Antibody, CR3022), and anti-Tubulin (Sigma, T6199). HRP-conjugated secondary antibodies (anti-mouse or anti-rabbit) were purchased from Amersham. Alexa Fluor fluorescent secondary antibodies were purchased from Invitrogen.
3
Antibodies for Immune Signaling Analysis
4
Immunoblot and Immunoprecipitation of STING Signaling
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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).
5
Immunoprecipitation and Immunoblotting Analysis
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Cell lysates were prepared in lysis buffer containing 1% Nonidet P-40 and protease inhibitor cocktail (Roche) (Cao et al., 2003 (link)). Soluble proteins were immunoprecipitated using anti-Flag (M2, Sigma), anti-Myc (Sigma), or IgG of the same isotype from the same species as a negative control (Sigma). An aliquot of the total lysate (5%, vol/vol) was included as a control. Immunoblotting was performed with horseradish peroxidase (HRP)-conjugated anti-Myc (Sigma), HRP-conjugated anti-Flag (Sigma), HRP-conjugated anti-β-actin (Sigma), anti-VP35 (Creative Diagnostics), anti-IRF3 (Cell Signaling Technology), anti-phospho-IRF3 Ser396 (Cell Signaling Technology), anti-STING (Proteintech), or anti-NP (Sino Biological) antibodies. The antigen–antibody complexes were visualized via chemiluminescence (Immobilon Western Chemiluminescent HRP Substrate, Millipore). A PageRuler Western marker (Thermo) was used as a molecular weight standard.
6
Proteomic Analysis of Colon Tissue Fractions
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Colon tissues, cells, and EVs fractions were homogenized using the RIPA buffer (Jiangsu KeyGEN BioTECH, Nanjing, China), phosphatase inhibitor cocktail, and protease inhibitor cocktail (Jiangsu KeyGEN BioTECH, Nanjing, China). Pierce BCA Protein Assay Kit (Jiangsu KeyGEN BioTECH, Nanjing, China) was used to determine the protein concentration. Anti-CD63 (A5271, ABclonal), anti-Alix (12422-1-AP, Proteintech), anti-CD81 (66866-1-Ig, Proteintech), anti-Calnexin (ab22595, Abcam), anti-STING (19851-1-AP, Proteintech), anti-Phospho-STING (Ser366) (85735, Cell Signaling Technology), anti-Phospho-STING (Ser365) (72971, Cell Signaling Technology), anti-IRF3 (4302, Cell Signaling Technology), anti-Phospho-IRF3 (Ser396) (29047, Cell Signaling Technology), anti-NF-kB p65 (A19653, ABclonal), anti-Phospho-NF-kB p65 (Ser536) (3303, Cell Signaling Technology), anti-Beta-Actin (4970, Cell Signaling Technology), anti-GAPDH (AF1186, Beyotime Biotechnology) and secondary antibodies (KGP1201, Jiangsu KeyGEN BioTECH, Nanjing, China) were used for western blot.
7
Cell Culture and Antibody Application
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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)
8
Western Blot Analysis of Immune Signaling
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Lysates were prepared in lysis buffer (1x SDS Sample Buffer, 62.5 mM Tris-HCl pH 6.8, 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue). Proteins were resolved by standard 10% SDS-PAGE and electroblotted onto nitrocellulose membranes. Membranes were blocked with 4% bovine serum albumin (w/v) in TBST and protein bands were detected with specific antibodies using chemiluminescence reagents and a G:BOX Chemi XRQ chemiluminescence imager (Syngene). The following rabbit antibodies were used: anti-phospho IRF3 (Ser 396) (1:1000; Cell Signaling #4947), anti-phospho-TBK1 (Ser 172) (1:1000; Cell Signaling #5483), and anti-ISG15 (1:1000; Cell Signaling #9636). Immunoreactive bands were visualized by incubation with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulins (1:5000) or goat anti-mouse immunoglobulins (1:1000; Bio-Rad). To ensure that equal amounts of proteins were loaded, blots were re-probed with α-tubulin (1:3000; Sigma-Aldrich). To detect multiple proteins, membranes were re-probed after stripping of previously used antibodies using a pH 2.2 glycine-HCl/SDS buffer.
9
Analyzing Immune Response Signaling Pathways
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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).
10
Phosphorylation Dynamics of NF-κB and IRF3
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mBMDC cells (5×105 cells/mL) cells were rested overnight and then treated with 5 µM 2D216 in the presence or absence of 1 ng/mL LPS for indicated time periods. Treated cells were lysed with PhosphoSafe Extraction Reagent (71296, EMD Millipore, Billerica, MA, United States) mixed with protease inhibitor cocktail (11697498001, Roche, Manheim, Germany) and 0.1% SDS. Protein concentrations were determined using Pierce BCA protein assay kit (23225, Thermo Fisher Scientific). The reduced and denatured cell lysates (10 µg protein) were separated by gel electrophoresis and transferred onto PVDF membranes. The membranes were blocked with 5% bovine serum albumin (BSA) in 0.1% Tween-20 Tris-buffered saline (TBST). The membranes were incubated in primary antibody and secondary antibody diluted in 5% - BSA- TBST and signals were visualized with SuperSignal™ West Dura Extended Duration Substrate (34075, Thermo Fisher Scientific). Anti-phospho NF-κB p65, and anti-phospho IRF3 (Ser396) and anti-actin were purchased from Cell Signaling Technology (Danvers, MA).
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