Product: Phospho-IRF3 (Ser396) Antibody
Catalog: AF2436
Description: Rabbit polyclonal antibody to Phospho-IRF3 (Ser396)
Application: WB IHC IF/ICC
Reactivity: Human, Mouse, Rat
Prediction: Pig, Bovine, Horse, Sheep
Mol.Wt.: 45-55kDa; 47kD(Calculated).
Uniprot: Q14653
RRID: AB_2845449

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 100ul $280 In stock
 200ul $350 In stock

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Product Info

Source:
Rabbit
Application:
WB 1:500-1:2000, IHC 1:50-1:200, IF/ICC 1:100-1:500
*The optimal dilutions should be determined by the end user.
*Tips:

WB: For western blot detection of denatured protein samples. IHC: For immunohistochemical detection of paraffin sections (IHC-p) or frozen sections (IHC-f) of tissue samples. IF/ICC: For immunofluorescence detection of cell samples. ELISA(peptide): For ELISA detection of antigenic peptide.

Reactivity:
Human,Mouse,Rat
Prediction:
Pig(100%), Bovine(88%), Horse(100%), Sheep(88%)
Clonality:
Polyclonal
Specificity:
Phospho-IRF3 (Ser396) Antibody detects endogenous levels of IRF3 only when phosphorylated at Ser396.
RRID:
AB_2845449
Cite Format: Affinity Biosciences Cat# AF2436, RRID:AB_2845449.
Conjugate:
Unconjugated.
Purification:
The antibody is from purified rabbit serum by affinity purification via sequential chromatography on phospho-peptide and non-phospho-peptide affinity columns.
Storage:
Rabbit IgG in phosphate buffered saline , pH 7.4, 150mM NaCl, 0.02% sodium azide and 50% glycerol. Store at -20 °C. Stable for 12 months from date of receipt.
Alias:

Fold/Unfold

IIAE7; Interferon regulatory factor 3; IRF 3; IRF-3; IRF3; IRF3_HUMAN; MGC94729;

Immunogens

Immunogen:
Uniprot:
Gene(ID):
Expression:
Q14653 IRF3_HUMAN:

Expressed constitutively in a variety of tissues.

Sequence:
MGTPKPRILPWLVSQLDLGQLEGVAWVNKSRTRFRIPWKHGLRQDAQQEDFGIFQAWAEATGAYVPGRDKPDLPTWKRNFRSALNRKEGLRLAEDRSKDPHDPHKIYEFVNSGVGDFSQPDTSPDTNGGGSTSDTQEDILDELLGNMVLAPLPDPGPPSLAVAPEPCPQPLRSPSLDNPTPFPNLGPSENPLKRLLVPGEEWEFEVTAFYRGRQVFQQTISCPEGLRLVGSEVGDRTLPGWPVTLPDPGMSLTDRGVMSYVRHVLSCLGGGLALWRAGQWLWAQRLGHCHTYWAVSEELLPNSGHGPDGEVPKDKEGGVFDLGPFIVDLITFTEGSGRSPRYALWFCVGESWPQDQPWTKRLVMVKVVPTCLRALVEMARVGGASSLENTVDLHISNSHPLSLTSDQYKAYLQDLVEGMDFQGPGES

Predictions

Predictions:

Score>80(red) has high confidence and is suggested to be used for WB detection. *The prediction model is mainly based on the alignment of immunogen sequences, the results are for reference only, not as the basis of quality assurance.

Species
Results
Score
Pig
100
Horse
100
Bovine
88
Sheep
88
Dog
75
Xenopus
0
Zebrafish
0
Chicken
0
Rabbit
0
Model Confidence:
High(score>80) Medium(80>score>50) Low(score<50) No confidence

PTMs - Q14653 As Substrate

Site PTM Type Enzyme
T3 Phosphorylation
S14 Phosphorylation
K29 Ubiquitination
K70 Sumoylation
K70 Ubiquitination
T75 Phosphorylation
K77 Ubiquitination
K87 Sumoylation
K87 Ubiquitination
S97 Phosphorylation
S123 Phosphorylation
T135 Phosphorylation P78527 (PRKDC)
S173 Phosphorylation P45984 (MAPK9) , P45983 (MAPK8) , Q9UHD2 (TBK1)
S175 Phosphorylation Q9UHD2 (TBK1)
T180 Phosphorylation
S188 Phosphorylation
K193 Ubiquitination
T237 Phosphorylation
T244 Phosphorylation
T253 Phosphorylation
S259 Phosphorylation
K313 Ubiquitination
K315 Ubiquitination
S339 Phosphorylation
T370 Phosphorylation
S385 Phosphorylation Q9UHD2 (TBK1)
S386 Phosphorylation Q14164 (IKBKE) , Q9UQM7 (CAMK2A) , Q9UHD2 (TBK1)
T390 Phosphorylation
S396 Phosphorylation O14920 (IKBKB) , Q14164 (IKBKE) , Q9UHD2 (TBK1)
S398 Phosphorylation O14920 (IKBKB) , Q14164 (IKBKE) , Q9UHD2 (TBK1)
S402 Phosphorylation O14920 (IKBKB) , Q14164 (IKBKE) , Q9UHD2 (TBK1)
T404 Phosphorylation O14920 (IKBKB) , Q9UHD2 (TBK1)
S405 Phosphorylation O14920 (IKBKB) , Q9UHD2 (TBK1)
S427 Phosphorylation

Research Backgrounds

Function:

Key transcriptional regulator of type I interferon (IFN)-dependent immune responses which plays a critical role in the innate immune response against DNA and RNA viruses. Regulates the transcription of type I IFN genes (IFN-alpha and IFN-beta) and IFN-stimulated genes (ISG) by binding to an interferon-stimulated response element (ISRE) in their promoters. Acts as a more potent activator of the IFN-beta (IFNB) gene than the IFN-alpha (IFNA) gene and plays a critical role in both the early and late phases of the IFNA/B gene induction. Found in an inactive form in the cytoplasm of uninfected cells and following viral infection, double-stranded RNA (dsRNA), or toll-like receptor (TLR) signaling, is phosphorylated by IKBKE and TBK1 kinases. This induces a conformational change, leading to its dimerization and nuclear localization and association with CREB binding protein (CREBBP) to form dsRNA-activated factor 1 (DRAF1), a complex which activates the transcription of the type I IFN and ISG genes. Can activate distinct gene expression programs in macrophages and can induce significant apoptosis in primary macrophages.

PTMs:

Constitutively phosphorylated on many Ser/Thr residues. Activated following phosphorylation by TBK1 and IKBKE. Innate adapter protein MAVS, STING1 or TICAM1 are first activated by viral RNA, cytosolic DNA, and bacterial lipopolysaccharide (LPS), respectively, leading to activation of the kinases TBK1 and IKBKE. These kinases then phosphorylate the adapter proteins on the pLxIS motif, leading to recruitment of IRF3, thereby licensing IRF3 for phosphorylation by TBK1. Phosphorylated IRF3 dissociates from the adapter proteins, dimerizes, and then enters the nucleus to induce IFNs.

(Microbial infection) Phosphorylation and subsequent activation of IRF3 is inhibited by vaccinia virus protein E3.

Ubiquitinated; ubiquitination involves RBCK1 leading to proteasomal degradation. Polyubiquitinated; ubiquitination involves TRIM21 leading to proteasomal degradation.

ISGylated by HERC5 resulting in sustained IRF3 activation and in the inhibition of IRF3 ubiquitination by disrupting PIN1 binding. The phosphorylation state of IRF3 does not alter ISGylation.

Subcellular Location:

Cytoplasm. Nucleus.
Note: Shuttles between cytoplasmic and nuclear compartments, with export being the prevailing effect (PubMed:10805757). When activated, IRF3 interaction with CREBBP prevents its export to the cytoplasm (PubMed:10805757).

Extracellular region or secreted Cytosol Plasma membrane Cytoskeleton Lysosome Endosome Peroxisome ER Golgi apparatus Nucleus Mitochondrion Manual annotation Automatic computational assertionSubcellular location
Tissue Specificity:

Expressed constitutively in a variety of tissues.

Subunit Structure:

Monomer. Homodimer; phosphorylation-induced. Interacts (when phosphorylated) with CREBBP. Interacts with MAVS (via phosphorylated pLxIS motif). Interacts with TICAM1 (via phosphorylated pLxIS motif). Interacts with STING1 (via phosphorylated pLxIS motif). Interacts with IKBKE and TBK1. Interacts with TICAM2. Interacts with RBCK1. Interacts with HERC5. Interacts with DDX3X (phosphorylated at 'Ser-102'); the interaction allows the phosphorylation and activation of IRF3 by IKBKE. Interacts with TRIM21 and ULK1, in the presence of TRIM21; this interaction leads to IRF3 degradation by autophagy. Interacts with RIOK3; RIOK3 probably mediates the interaction of TBK1 with IRF3. Interacts with ILRUN; the interaction inhibits IRF3 binding to its DNA consensus sequence. Interacts with LYAR; this interaction impairs IRF3 DNA-binding activity.

(Microbial infection) Interacts with rotavirus A NSP1 (via pLxIS motif); this interaction leads to the proteasome-dependent degradation of IRF3.

(Microbial infection) Interacts with herpes virus 8/HHV-8 protein VIRF1.

(Microbial infection) Interacts with Seneca Valley virus protease 3C; this interaction is involved in the suppression of IRF3 expression and phosphorylation by the virus.

Family&Domains:

Belongs to the IRF family.

Research Fields

· Human Diseases > Infectious diseases: Bacterial > Pertussis.

· Human Diseases > Infectious diseases: Viral > Hepatitis C.

· Human Diseases > Infectious diseases: Viral > Hepatitis B.

· Human Diseases > Infectious diseases: Viral > Measles.

· Human Diseases > Infectious diseases: Viral > Influenza A.

· Human Diseases > Infectious diseases: Viral > Human papillomavirus infection.

· Human Diseases > Infectious diseases: Viral > Herpes simplex infection.

· Human Diseases > Infectious diseases: Viral > Epstein-Barr virus infection.

· Human Diseases > Cancers: Overview > Viral carcinogenesis.

· Organismal Systems > Immune system > Toll-like receptor signaling pathway.   (View pathway)

· Organismal Systems > Immune system > NOD-like receptor signaling pathway.   (View pathway)

· Organismal Systems > Immune system > RIG-I-like receptor signaling pathway.   (View pathway)

· Organismal Systems > Immune system > Cytosolic DNA-sensing pathway.   (View pathway)

References

1). iNOS aggravates pressure overload-induced cardiac dysfunction via activation of the cytosolic-mtDNA-mediated cGAS-STING pathway. Theranostics, 2023 (PubMed: 37554263) [IF=12.4]

Application: WB    Species: Mouse    Sample: heart tissues

Figure 1 iNOS contributes to cytosolic mtDNA accumulation and cGAS activation in pressure overload-stressed heart. (A) Representative co-immunostaining of dsDNA and mitochondria (Tom20) showing cytosolic DNA in heart tissues. (B) Cytosolic mtDNA content in the hearts of TAC mice were measured using quantitative-PCR, n = 4. (C) Total mtDNA in the hearts of TAC mice, n = 4. (D-E) iNOS deficiency decreased cGAS-STING expression and IRF-3 phosphorylation in the hearts of TAC mice, n = 6. (F-G) iNOS deficiency reduced the mRNA expression levels of IFN-β and ISG activated by cGAS-STING in TAC hearts, n = 4. Data are presented as the mean ± SEM. Statistical analysis was performed using a 1 or 2-way ANOVA with a Tukey's multiple-comparison post-hoc test comparisons between multiple groups. TAC, transverse aortic constriction; iNOS. inducible NO synthase; mtDNA, mitochondrial DNA; dsDNA, double-stranded DNA; cGAS, cyclic GMP-AMP synthase; STING, stimulator of interferon genes; IFN-β, interferon-β.

2). A brain-enriched lncRNA shields cancer cells from immune-mediated killing for metastatic colonization in the brain. Proceedings of the National Academy of Sciences, 2022 (PubMed: 35617432) [IF=11.1]

3). Commensal cow Roseburia reduces gut-dysbiosis-induced mastitis through inhibiting bacterial translocation by producing butyrate in mice. Cell Reports, 2022 (PubMed: 36417859) [IF=8.8]

4). Targeting the smooth muscle cell KEAP1-Nrf2-STING axis with pterostilbene attenuates abdominal aortic aneurysm. Phytomedicine : international journal of phytotherapy and phytopharmacology, 2024 (PubMed: 38763007) [IF=7.9]

5). Chronic low-grade inflammation is involved in TLR4 knockout-induced spontaneous obesity in aged mice. BIOMEDICINE & PHARMACOTHERAPY, 2022 (PubMed: 35093760) [IF=7.5]

Application: WB    Species: Mouse    Sample: liver

Fig. 5. TLR4 KO activated the TRIF pathway in the liver. a mRNA expression of TRIF and IRF3, n = 6. b Protein expression of hepatic TRIF, IRF3 and pIRF3, and the relative TRIF, IRF3 and pIRF3 protein levels is shown in bar graph. c Immunoreactivities of pIRF3 were detected by immunohistochemistry and relative optical density of immunoreactivities, n = 6. Scale bar is 50 µm. Data are presented as mean ± S.E.M. *P 

6). Role of the cGAS-STING Pathway in Aging-related Endothelial Dysfunction. Aging and Disease, 2022 (PubMed: 36465181) [IF=7.4]

Application: WB    Species: Mouse    Sample:

Figure 1. Age-dependent changes in vasodilation function, and eNOS, p53, p21 p16 cGAS, STING and p-IRF3/IRF3 expression levels in mice. Acetylcholine (ACh)-induced relaxation (A) and sodium nitroprusside (SNP)-induced relaxation (B) of mouse aortas were measured in the 2 Months, 6 Months, 12 Months and 24 Months age groups. (C) The protein levels of eNOS, p53, p21, p16, cGAS, STING, p-IRF3, IRF3 and β-actin were measured in the aortas of mice in each age group by western blot analysis. β-actin was used as the housekeeper protein for normalization. Quantification of protein levels is shown in (D). Data were analyzed by (A and B) two-way ANOVA or (D) one way ANOVA plus Bonferroni post hoc test. All data shown are mean±SD. AU indicates arbitrary units. Relative expression is the fold changes relative to the 2 Months group. n=6, *P

7). The PICK1/TLR4 complex on microglia is involved in the regulation of LPS-induced sepsis-associated encephalopathy. International Immunopharmacology, 2021 (PubMed: 34500285) [IF=5.6]

Application: WB    Species: Mouse    Sample:

Fig. 2. After LPS stimulation, the combination of TLR4 and IBa1 increases, and the TLR4 signaling pathway is activated. (A) Representative images show cellular localization of TLR4 in microglia in the hippocampus, Scale bar, 100 μm.Look at the red box and zoom in and you can see that: blue staining represents DAPI, red staining represents IBa1, green staining represents TLR4, Scale bar, 5 μm (B) Representative images of Western blots of LPS-treated mice of Hippocampus. (C–H) Quantitative analyses of IBa1, TLR4, TRAF3, phospho-TBK1, phospho-IKKε, and phospho-IRF3.The data are expressed as the mean ± standard error of the mean

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