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8348
Rig-I Pathway Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Rig-I Pathway Antibody Sampler Kit #8348

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Rig-I Pathway Antibody Sampler Kit: Image 1

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with human MDA-5 (+), using MDA-5 (D74E4) Rabbit mAb.

Rig-I Pathway Antibody Sampler Kit: Image 2

Western blot analysis of differentiated THP-1 and RAW 264.7 cells, untreated or LPS-treated (1 ug/ml, overnight), using Rig-I (D14G6) Rabbit mAb.

Rig-I Pathway Antibody Sampler Kit: Image 3

Confocal immunofluorescent analysis of MCF-7 cells using MAVS Antibody (green) showing colocalization with mitochondria that have been labeled with MitoTracker® Red CMXRos (red). Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).

Rig-I Pathway Antibody Sampler Kit: Image 4

Western blot analysis of extracts from control HeLa cells (lane 1) or IRF-3 knockout HeLa cells (lane 2) using IRF-3 (D6I4C) XP® Rabbit mAb, #11904 (upper) or β-actin (13E5) Rabbit mAb, #4970 (lower). The absence of signal in the IRF-3 knockout HeLa cells confirms specificity of the antibody for IRF-3.

Rig-I Pathway Antibody Sampler Kit: Image 5

Confocal immunofluorescent analysis of HT-29 cells, untransfected (left) or transfected with Poly(I:C) (2.5 μg/ml, 6 hr; right), using IRF-3 (D6I4C) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Rig-I Pathway Antibody Sampler Kit: Image 6

Western blot analysis of HCT116 cell extracts, untreated (-) or TBK1/NAK knock-out (+), using TBK/NAK (D1B4) Rabbit mAb Antibody #3504 (upper) or β-actin (D6A8) Rabbit mAb #8457 (lower).

Rig-I Pathway Antibody Sampler Kit: Image 7

Flow cytometric analysis of THP-1 cells differentiated with TPA (80nM, 4 days) #9905, untreated (blue) or treated with LPS (1 ng/mL, 1 hr; green) #14011 using Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

Rig-I Pathway Antibody Sampler Kit: Image 8

Western blot analysis of extracts from HT29 and THP1 cells, control or plpC-transfected (1 hour), using Phospho-IRF-3 (Ser396) (4D4G) Rabbit mAb.

Rig-I Pathway Antibody Sampler Kit: Image 9

Western blot analysis of extracts from THP-1 cells, differentiated with TPA #4174 (80 nM, overnight) followed by treatment with LPS (1 μg/ml, indicated times), using Phospho-IKKε (Ser172) (D1B7) Rabbit mAb (upper) or IKKε (D20G4) Rabbit mAb #2905 (lower).

Rig-I Pathway Antibody Sampler Kit: Image 10

Western blot analysis of recombinant GST-IKKε #7553 and lysates from Ramos and RL cells using IKKε (D20G4) Rabbit mAb.

Rig-I Pathway Antibody Sampler Kit: Image 11

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

Rig-I Pathway Antibody Sampler Kit: Image 12

Western blot anlaysis of extracts from differentiated THP-1 cells, untreated or treated with LPS (1 μg/ml, indicated times), using MDA-5 (D74E4) Rabbit mAb.

Rig-I Pathway Antibody Sampler Kit: Image 13

Western blot analysis of extracts from LNCaP, MCF-7, and HT29 cell lines using MAVS Antibody.

Rig-I Pathway Antibody Sampler Kit: Image 14

Immunoprecipitation of IRF-3 from THP-1 cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or IRF-3 (D6I4C) XP® Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using IRF-3 (D6I4C) XP® Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 and Anti-mouse IgG, HRP-linked Antibody #7076 were used as secondary antibodies.

Rig-I Pathway Antibody Sampler Kit: Image 15

Western blot analysis of extracts from various cell lines using TBK1/NAK (D1B4) Rabbit mAb.

Rig-I Pathway Antibody Sampler Kit: Image 16

Confocal immunofluorescent analysis of THP-1 cells differentiated with TPA #4174 (80nM, overnight) (left), followed by treatment with LPS (1μg/ml, 1 hour) (center) or LPS with λ phosphatase treatment (right) using Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 Phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Rig-I Pathway Antibody Sampler Kit: Image 17

Western blot analysis of extracts from HeLa cells, mock-transfected or transfected with human MAVS, using MAVS Antibody.

Rig-I Pathway Antibody Sampler Kit: Image 18

Western blot analysis of extracts from various cell lines using IRF-3 (D6I4C) XP® Rabbit mAb.

Rig-I Pathway Antibody Sampler Kit: Image 19

Western blot analysis of extracts from THP-1 cells differentiated with TPA #4174 (80 nM, overnight) followed by treatment with LPS (1 μg/ml), up to 24h, using Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb (upper), or total TBK1/NAK (D1B4) Rabbit mAb #3504 (lower).

Rig-I Pathway Antibody Sampler Kit: Image 20

Western blot analysis of extracts from THP-1 cells differentiated with TPA #4174 (80 nM, overnight) followed by treatment with LPS (1 μg/ml, 1 hour), with or without phosphatase treatment using Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb (upper), or total TBK1/NAK (D1B4) Rabbit mAb #3504 (lower).

To Purchase # 8348T
Product # Size Price
8348T
1 Kit  (9 x 20 µl) $ 609

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
MDA-5 (D74E4) Rabbit mAb 5321 20 µl
  • WB
  • IP
H M 135 Rabbit IgG
Rig-I (D14G6) Rabbit mAb 3743 20 µl
  • WB
  • IP
H M R Mk 102 Rabbit IgG
MAVS Antibody 3993 20 µl
  • WB
  • IF
H 75, 52 Rabbit 
IRF-3 (D6I4C) XP® Rabbit mAb 11904 20 µl
  • WB
  • IP
  • IF
H Mk 50-55 Rabbit IgG
TBK1/NAK (D1B4) Rabbit mAb 3504 20 µl
  • WB
  • IP
H M R Mk 84 Rabbit 
Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb 5483 20 µl
  • WB
  • IP
  • IF
  • F
H M 84 Rabbit IgG
Phospho-IRF-3 (Ser396) (4D4G) Rabbit mAb 4947 20 µl
  • WB
H M 45-55 Rabbit IgG
Phospho-IKKε (Ser172) (D1B7) Rabbit mAb 8766 20 µl
  • WB
  • IP
H 80 Rabbit IgG
IKKε (D20G4) Rabbit mAb 2905 20 µl
  • WB
  • IP
H 80 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product 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 two western blot experiments per antibody.

Specificity / Sensitivity

MDA-5 (D74E4) Rabbit mAb, Rig-I (D14G6) Rabbit mAb, MAVS Antibody, IRF-3 (D6I4C) XP® Rabbit mAb, TBK1/NAK (D1B4) 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. This antibody may cross-react with phospho-IKKε. Phospho-IRF-3 (Ser396) (4D4G) Rabbit mAb detects endogenous levels of IRF-3 only when phosphorylated at Ser396. Phospho-IKKε (Ser172) (D1B7) Rabbit mAb recognizes endogenous levels of IKKε protein only when phosphorylated at Ser172. This antibody may cross-react with phospho-TBK1/NAK.

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, Ser645 of human TBK1/NAK protein, Val345 of human IKKε protein, or recombinant human IRF-3 protein. Activation state monoclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser172 of human TBK1/NAK protein, Ser396 of human IRF-3 protein, or Ser172 of human IKKε 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.

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