Revision 1

#40415Store at -20C

1 Kit

(9 x 20 microliters)

Cell Signaling Technology

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

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
TBK1/NAK (E8I3G) Rabbit mAb 38066 20 µl 84 kDa Rabbit IgG
MAVS (D5A9E) Rabbit mAb 24930 20 µl 75, 52 kDa Rabbit IgG
TRAF2 (C192) Antibody 4724 20 µl 53 kDa Rabbit 
TRAF3 (E8H3B) Rabbit mAb 33640 20 µl 62 kDa Rabbit IgG
TRAF6 (E2K9D) Rabbit mAb 67591 20 µl 60 kDa Rabbit IgG
Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb 5483 20 µl 84 kDa Rabbit IgG
IRF-3 (D6I4C) XP® Rabbit mAb 11904 20 µl 50-55 kDa Rabbit IgG
Phospho-IRF-3 (Ser386) (E7J8G) XP® Rabbit mAb 37829 20 µl 50-55 kDa Rabbit IgG
Phospho-IRF-3 (Ser396) (D6O1M) Rabbit mAb 29047 20 µl 45-55 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 MAVS Signalosome Antibody Sampler Kit provides an economical means of detecting the activation of signaling pathways in which MAVS plays a critical role. The kit includes enough antibodies to perform two western blot experiments with each primary 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 antibodies.

Background

Recognition of conserved molecular structures of viruses by host pattern-recognition receptors (PRRs) initiates innate antiviral immune responses. Several families of PRRs have been demonstrated to sense different microbial components (1,2). The RIG-I-like receptor (RLR) family, including RIG-I, MDA5, and LGP2, are the primary PRRs to recognize viral RNAs (3,4). Upon binding to viral RNA, these receptors undergo conformation change, leading to their interaction with mitochondrial antiviral signaling protein (MAVS). MAVS subsequently forms large prion-like polymers and serves as a platform to recruit multiple components, including TRAF proteins and TBK1, to form the so-called MAVS signalosome. MAVS signalosome, in turn, activates the IRF-3 and NF-kB pathways, leading to the production of type I IFNs and pro-inflammatory cytokines (5-9).

  1. Akira, S. et al. (2006) Cell 124, 783-801.
  2. Kumar, H. et al. (2011) Int Rev Immunol 30, 16-34.
  3. Nakhaei, P. et al. (2009) Semin Immunol 21, 215-22.
  4. Yoneyama, M. et al. (2015) Curr Opin Immunol 32, 48-53.
  5. Seth, R.B. et al. (2005) Cell 122, 669-82.
  6. Vazquez, C. and Horner, S.M. (2015) J Virol 89, 6974-7.
  7. Reikine, S. et al. (2014) Front Immunol 5, 342.
  8. Dutta, S. et al. (2020) Front Microbiol 11, 1990.
  9. Thoresen, D. et al. (2021) Immunol Rev 304, 154-168.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    XP is a registered 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

    Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

    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.

    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.
    Revision 1
    #40415

    MAVS Signalosome Antibody Sampler Kit

    MAVS Signalosome 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.
    MAVS Signalosome Antibody Sampler Kit: Image 2 Expand Image
    Western blot analysis of extracts from HT-29 cells, untreated or treated with h-IFNa (10 ng/mL, 16 hr), followed by poly(I:C) (2.5 mg/mL, 7 hr) and MG-132 (#2194, 10mM, 7 hr) using Phospho-IRF-3 (Ser386) (E7J8G) XP® Rabbit mAb #37829 (upper), IRF-3 (D6I4C) XP® Rabbit mAb #11904 (middle), and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
    MAVS Signalosome Antibody Sampler Kit: Image 3 Expand Image
    Simple Western™ analysis of lysates (0.1 mg/mL) from 293T cells using TRAF2 (C192) Antibody #4724. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (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.
    MAVS Signalosome Antibody Sampler Kit: Image 4 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.
    MAVS Signalosome Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing full-length human MAVS (hMAVS; +), using MAVS (D5A9E) Rabbit mAb.
    MAVS Signalosome Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of adipocytes from wild type (WT) mice, untransfected (-) or transfected with Poly (I:C) (2.5 μg/ml, 6 hr; +), and adipocytes from IRF-3 (-/-) mice, untransfected (-) or transfected with Poly (I:C) (2.5 μg/ml, 6 hr; +), using Phospho-IRF-3 (Ser396) (D6O1M) Rabbit mAb (upper), IRF-3 (D83B9) Rabbit mAb #4302 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).
    MAVS Signalosome Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from various cell lines using TRAF3 (E8H3B) Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). RPMI 8226 cells harbor a heterozygous mutation in the TRAF3 gene resulting in loss of expression. Absence of signal in RPMI 8226 cells confirms the specificity of the antibody.
    MAVS Signalosome Antibody Sampler Kit: Image 8 Expand Image
    Immunoprecipitation of TRAF3 protein from TF-1 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is TRAF3 (E8H3B) Rabbit mAb. Western blot analysis was performed using TRAF3 (E8H3B) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.
    MAVS Signalosome Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from A549 cells, untreated (-) or treated with Poly (I:C) (+), using Phospho-IRF-3 (Ser386) (E7J8G) XP® Rabbit mAb (upper) or IRF-3 (D6I4C) XP® Rabbit mAb #11904 (lower).
    MAVS Signalosome Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from HCT 116 cells, either wild-type (+/+) or TBK1/NAK knockout (-/-), using TBK1/NAK (E8I3G) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
    MAVS Signalosome Antibody Sampler Kit: Image 11 Expand Image
    Western blot analysis of extracts from Jurkat, 293, HeLa, C2C12, and NIH/3T3 cells, using TRAF2 (C192) Antibody.
    MAVS Signalosome Antibody Sampler Kit: Image 12 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).
    MAVS Signalosome Antibody Sampler Kit: Image 13 Expand Image
    Western blot analysis of extracts from varous cell lines using TRAF6 (E2K9D) Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). Absence of signal in NCI-H23 cells is predicted by RNAseq and confirms the specificity of the antibody.
    MAVS Signalosome Antibody Sampler Kit: Image 14 Expand Image
    Immunoprecipitation of TRAF6 protein from K-562 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is TRAF6 (E2K9D) Rabbit mAb. Western blot analysis was performed using TRAF6 (E2K9D) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.
    MAVS Signalosome Antibody Sampler Kit: Image 15 Expand Image
    Immunoprecipitation of TRAF6 protein from A20 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is TRAF6 (E2K9D) Rabbit mAb. Western blot analysis was performed using TRAF6 (E2K9D) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.
    MAVS Signalosome Antibody Sampler Kit: Image 16 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.
    MAVS Signalosome Antibody Sampler Kit: Image 17 Expand Image
    Western blot analysis of extracts from various cell lines using IRF-3 (D6I4C) XP® Rabbit mAb.
    MAVS Signalosome Antibody Sampler Kit: Image 18 Expand Image
    Western blot analysis of extracts from various cell lines using MAVS (D5A9E) Rabbit mAb.
    MAVS Signalosome Antibody Sampler Kit: Image 19 Expand Image
    Western blot analysis of HT-29 cells, untransfected (-) or transfected with Poly (I:C) (2.5 μg/ml, 6 hr; +), using Phospho-IRF-3 (Ser396) (D6O1M) Rabbit mAb (upper) or IRF-3 (D6I4C) XP® Rabbit mAb #11904 (lower).
    MAVS Signalosome Antibody Sampler Kit: Image 20 Expand Image
    Confocal immunofluorescent analysis of THP-1 cells, untransfected (left) or transfected with Poly (I:C) (2.5 μg/ml, 6 hr; right), using Phospho-IRF-3 (Ser386) (E7J8G) XP® Rabbit mAb (green) and β-Actin (13E5) Rabbit mAb (Alexa Fluor® 647 Conjugate) #8584 (red). Blue = Hoechst 33342 #4082 (fluorescent DNA dye).
    MAVS Signalosome Antibody Sampler Kit: Image 21 Expand Image
    Western blot analysis of extracts from various cell lines using TBK1/NAK (E8I3G) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
    MAVS Signalosome Antibody Sampler Kit: Image 22 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).
    MAVS Signalosome Antibody Sampler Kit: Image 23 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.
    MAVS Signalosome Antibody Sampler Kit: Image 24 Expand Image
    Immunoprecipitation of MAVS protein from MCF7 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is MAVS (D5A9E) Rabbit mAb. Western blot was performed using MAVS (D5A9E) Rabbit mAb.
    MAVS Signalosome Antibody Sampler Kit: Image 25 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 Phospho-IRF-3 (Ser396) (D6O1M) Rabbit mAb (green) and EpCAM (VU1D9) Mouse mAb (Alexa Fluor® 555 Conjugate) #5488 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    MAVS Signalosome Antibody Sampler Kit: Image 26 Expand Image
    Flow cytometric analysis of THP-1 cells differentiated with TPA #4174 (80 nM, 24 hr), and then untransfected (blue) or transfected with poly(dA:dT) (5 ug/mL, 3 hr; green), using Phospho-IRF-3 (Ser 386) (E7J8G) 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.
    MAVS Signalosome Antibody Sampler Kit: Image 27 Expand Image
    Immunoprecipitation of TBK1/NAK from Raji cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is TBK1/NAK (E8I3G) Rabbit mAb. Western blot analysis was performed using TBK1/NAK (E8I3G) Rabbit mAb.
    MAVS Signalosome Antibody Sampler Kit: Image 28 Expand Image
    Western blot analysis of U20S extracts from WT (left) or TBK1 KO (right) using TBK1/NAK (E8I3G) Rabbit mAb. Membranes stained with Ponceau S for total protein normalization (lower). These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibodies, as a companion to validation data generated by CST scientists.
    MAVS Signalosome Antibody Sampler Kit: Image 29 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).
    MAVS Signalosome Antibody Sampler Kit: Image 30 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).
    MAVS Signalosome Antibody Sampler Kit: Image 31 Expand Image
    Confocal immunofluorescent analysis of MCF7 cells showing colocalization of MAVS (D5A9E) Rabbit mAb (left, green) with the mitochondrial dye MitoTracker® Red CMXRos #9082 (middle, red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    MAVS Signalosome Antibody Sampler Kit: Image 32 Expand Image
    Flow cytometric analysis of adipocytes from wild type mice, untransfected (A) or transfected with Poly (I:C) (2.5 µg/ml, 6 hr; B), and adipocytes from IRF-3 (-/-) mice, untransfected (C) or transfected with Poly (I:C) (2.5 µg/ml, 6 hr; D), using Phospho-IRF-3 (Ser396) (D6O1M) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
    MAVS Signalosome Antibody Sampler Kit: Image 33 Expand Image
    Confocal immunofluorescent analysis of HCT 116 cells, wild-type (left, positive) or TBK1/NAK knockout (right, negative), using TBK1/NAK (E8I3G) Rabbit mAb (green). Actin filaments were labeled with Alexa Fluor® 555 Phalloidin #8953 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
    MAVS Signalosome Antibody Sampler Kit: Image 34 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.
    MAVS Signalosome Antibody Sampler Kit: Image 35 Expand Image
    Flow cytometric analysis of THP-1 cells (green) using MAVS (D5A9E) Rabbit mAb (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed line). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
    MAVS Signalosome Antibody Sampler Kit: Image 36 Expand Image
    Flow cytometric analysis of HT-29 cells, untransfected (blue) or transfected with Poly (I:C) (2.5 μg/ml, 6 hr; green), using Phospho-IRF-3 (Ser396) (D6O1M) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
    MAVS Signalosome Antibody Sampler Kit: Image 37 Expand Image
    Flow cytometric analysis of TALL-1 cells (blue, negative) and Raji cells (green, positive) using TBK1/NAK (E8I3G) 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.
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    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.