Product Pathways - NF-kB Signaling

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Rig-I Pathway Antibody Sampler Kit #8348

Kit Includes	Quantity	Applications	Reactivity	MW (kDa)	Isotype
MDA-5 (D74E4) Rabbit mAb #5321	40 µl	W IP	H M	135	Rabbit IgG
Rig-I (D14G6) Rabbit mAb #3743	40 µl	W IP	H M R Mk	102	Rabbit IgG
MAVS Antibody #3993	40 µl	W IF-IC	H	75, 52	Rabbit
IRF-3 (D83B9) Rabbit mAb #4302	40 µl	W IP	H M R Mk	45-55	Rabbit IgG
TBK1/NAK (D1B4) Rabbit mAb #3504	40 µl	W IP	H M R Mk	84	Rabbit IgG
Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb #5483	40 µl	W IP IF-IC F	H (M) (R) (Mk) (X) (B) (Dg)	84	Rabbit IgG
Phospho-IRF-3 (Ser396) (4D4G) Rabbit mAb #4947	40 µl	W	H M	45-55	Rabbit IgG
IKKε (D61F9) XP® Rabbit mAb #3416	40 µl	W IP IF-IC F	M R	80	Rabbit
IKKε (D20G4) Rabbit mAb #2905	40 µl	W IP	H (Mk)	80	Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody #7074	100 µl				Goat

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  X=Xenopus  B=Bovine  Dg=Dog
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

MDA-5 (D74E4) Rabbit mAb, Rig-I (D14G6) Rabbit mAb, MAVS Antibody, IRF-3 (D83B9) Rabbit mAb, TBK1/NAK (D1B4) Rabbit mAb, IKKε (D61F9) XP^® Rabbit mAb, and IKKε (D20G4) Rabbit mAb detect endogenous levels of respective total proteins and do not cross-react with other proteins. Bands detected at 52 and 75 kDa by MAVS Antibody correlate with those described by Seth et al. (2005). Phospho-TBK1/NAK (Ser172) (D52C2) XP^® Rabbit mAb detects endogenous levels of TBK1/NAK only when phosphorylated at Ser172. Phospho-IRF-3 (Ser396) (4D4G) Rabbit mAb detects endogenous levels of IRF-3 only when phosphorylated at Ser396.

Description

The Rig-I Pathway Antibody Sampler Kit provides an economical means to evaluate the activation state and total protein levels of multiple members of the Rig-I pathway including Rig-I, MDA-5, MAVS, IRF-3, TBK1/NAK, and IKKε. The kit includes enough primary antibody to perform four western blot experiments per antibody.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues at the carboxy terminus of human MAVS protein. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg470 of human MDA-5 protein, Lys652 of human Rig-I protein, the carboxy terminus of human IRF-3 protein, Ser645 of human TBK1/NAK protein, the carboxy terminus of mouse IKKε protein, or Val345 of human IKKε protein. Activation state monoclonal antibodies are producted by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser172 of human TBK1/NAK protein or Ser396 of human IRF-3 protein.

Background

Antiviral innate immunity depends on the combination of parallel pathways triggered by virus detecting proteins in the Toll-like receptor (TLR) family and RNA helicases, such as Rig-I (retinoic acid-inducible gene I) and MDA-5 (melanoma differentiation-associated antigen 5), which promote the transcription of type I interferons (IFN) and antiviral enzymes (1-3). TLRs and helicase proteins contain sites that recognize the molecular patterns of different virus types, including DNA, single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), and glycoproteins. These antiviral proteins are found in different cell compartments; TLRs (i.e. TLR3, TLR7, TLR8, and TLR9) are expressed on endosomal membranes and helicases are localized to the cytoplasm. Rig-I expression is induced by retinoic acid, LPS, IFN, and viral infection (4,5). Both Rig-I and MDA-5 share a DExD/H-box helicase domain that detects viral dsRNA and two amino-terminal caspase recruitment domains (CARD) that are required for triggering downstream signaling (4-7). Rig-I binds both dsRNA and viral ssRNA that contains a 5'-triphosphate end not seen in host RNA (8,9). Though structurally related, Rig-I and MDA-5 detect a distinct set of viruses (10,11). The CARD domain of the helicases, which is sufficient to generate signaling and IFN production, is recruited to the CARD domain of the MAVS/VISA/Cardif/IPS-1 mitochondrial protein, which triggers activation of NF-κB, TBK1/IKKε, and IRF-3/IRF-7 (12-15).

1. Yoneyama, M. and Fujita, T. (2007) J Biol Chem 282, 15315-8.
2. Meylan, E. and Tschopp, J. (2006) Mol Cell 22, 561-9.
3. Thompson, A.J. and Locarnini, S.A. (2007) Immunol Cell Biol 85, 435-45.
4. Imaizumi, T. et al. (2002) Biochem Biophys Res Commun 292, 274-9.
5. Zhang, X. et al. (2000) Microb Pathog 28, 267-78.
6. Yoneyama, M. et al. (2005) J Immunol 175, 2851-8.
7. Yoneyama, M. et al. (2004) Nat Immunol 5, 730-7.
8. Hornung, V. et al. (2006) Science 314, 994-7.
9. Pichlmair, A. et al. (2006) Science 314, 997-1001.
10. Kato, H. et al. (2006) Nature 441, 101-5.
11. Childs, K. et al. (2007) Virology 359, 190-200.
12. Meylan, E. et al. (2005) Nature 437, 1167-72.
13. Xu, L.G. et al. (2005) Mol Cell 19, 727-40.
14. Kawai, T. et al. (2005) Nat Immunol 6, 981-8.
15. Seth, R.B. et al. (2005) Cell 122, 669-82.

Application References

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Protocols

2905:

Immunoprecipitation, Western Blotting

3416:

Flow, Immunofluorescence, Immunoprecipitation, Western Blotting

3504:

Immunoprecipitation, Western Blotting

3743:

Immunoprecipitation, Western Blotting

3993:

Immunofluorescence, Western Blotting

4302:

Immunoprecipitation, Western Blotting

4947:

Western Blotting

5321:

Immunoprecipitation, Western Blotting

5483:

Flow, Immunofluorescence, Immunoprecipitation, Western Blotting

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For Research Use Only. Not For Use In Diagnostic Procedures.