Revision 1

#8348Store at -20C

1 Kit

(9 x 20 microliters)

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

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For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
MDA-5 (D74E4) Rabbit mAb 5321 20 µl 135 kDa Rabbit IgG
Rig-I (D14G6) Rabbit mAb 3743 20 µl 102 kDa Rabbit IgG
MAVS Antibody 3993 20 µl 75, 52 kDa Rabbit 
IRF-3 (D6I4C) XP® Rabbit mAb 11904 20 µl 50-55 kDa Rabbit IgG
TBK1/NAK (D1B4) Rabbit mAb 3504 20 µl 84 kDa Rabbit IgG
Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb 5483 20 µl 84 kDa Rabbit IgG
Phospho-IRF-3 (Ser396) (4D4G) Rabbit mAb 4947 20 µl 45-55 kDa Rabbit IgG
Phospho-IKKε (Ser172) (D1B7) Rabbit mAb 8766 20 µl 80 kDa Rabbit IgG
IKKε (D20G4) Rabbit mAb 2905 20 µl 80 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

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.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

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.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

    Limited Uses

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    Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

    Revision 1
    #8348

    Rig-I Pathway Antibody Sampler Kit

    Rig-I Pathway Antibody Sampler Kit: Image 1 Expand Image
    Simple Western™ analysis of lysates (1 mg/mL) from HeLa cells using IRF-3 (D6I4C) XP ® Rabbit mAb #11904. The virtual lane view (left) shows the target band (as indicated) at 1:50 and 1:250 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    Rig-I Pathway Antibody Sampler Kit: Image 2 Expand Image
    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 3 Expand Image
    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 4 Expand Image
    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 5 Expand Image
    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 6 Expand Image
    Western blot analysis of extracts from LNCaP, MCF-7, and HT29 cell lines using MAVS Antibody.
    Rig-I Pathway Antibody Sampler Kit: Image 7 Expand Image
    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 8 Expand Image
    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 9 Expand Image
    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 10 Expand Image
    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 11 Expand Image
    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 12 Expand Image
    Western blot analysis of extracts from various cell lines using IRF-3 (D6I4C) XP® Rabbit mAb.
    Rig-I Pathway Antibody Sampler Kit: Image 13 Expand Image
    Western blot analysis of extracts from various cell lines using TBK1/NAK (D1B4) Rabbit mAb.
    Rig-I Pathway Antibody Sampler Kit: Image 14 Expand Image
    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 15 Expand Image
    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 16 Expand Image
    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).
    Rig-I Pathway Antibody Sampler Kit: Image 17 Expand Image
    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 18 Expand Image
    Western blot analysis of extracts from Raw264.7 and CHO cells using Rig-I (D14G6) Rabbit mAb.
    Rig-I Pathway Antibody Sampler Kit: Image 19 Expand Image
    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 20 Expand Image
    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 21 Expand Image
    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 22 Expand Image
    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.