Revision 1

#39433Store at -20C

1 Kit

(6 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
MyD88 (D80F5) Rabbit mAb 4283 20 µl 33 kDa Rabbit IgG
IRAK1 (D51G7) Rabbit mAb 4504 20 µl 78-105 kDa Rabbit IgG
IRAK2 Antibody 4367 20 µl 62 kDa Rabbit 
IRAK4 Antibody 4363 20 µl 55 kDa Rabbit 
Phospho-IRAK4 (Thr345/Ser346) (D6D7) Rabbit mAb 11927 20 µl 55 kDa Rabbit IgG
TRAF6 (E2K9D) Rabbit mAb 67591 20 µl 60 kDa Rabbit IgG
TBK1/NAK (E8I3G) Rabbit mAb 38066 20 µl 84 kDa Rabbit IgG
Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb 5483 20 µl 84 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 Myddosome Complex Antibody Sampler Kit provides an economical means of detecting the components of the myddosome complex using phospho-specific and control antibodies. 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

Toll-like receptors (TLRs) are a large family of so-called pattern recognition receptors (PRRs) that detect pathogen associated molecular patterns (PAMPs) and danger associated molecular patterns (DAMPs) (1,2). Upon activation, TLRs initiate two main signaling pathways through their C-terminal cytoplasmic Toll/Il-1 receptor (TIR) domain that couples with TIR domain-containing adaptors MyD88 and TRIF. The MyD88-dependent pathway is initiated by the formation of a large oligomeric protein complex termed myddosome. Myddosome is one of so-called supramolecular organizing centers (SMOCs), a signaling organelle that is common for PRRs in the innate immune system. Myddosome formation promotes IRAK4 activation, which in turn activates IRAK1 and later, IRAK2. TRAF6 is then recruited and activated through the binding sites within IRAKs. Activated TRAF6 is released to the cytosol and triggers the IKK complex to activate the NF-κB pathway to mediate the expression of pro-inflammatory cytokines and chemokines (3-7). Recently, it was also found that TBK1 is recruited to the myddosome complex and activated to induce aerobic glycolysis (8).

  1. Behzadi, P. et al. (2021) J Immunol Res 2021, 9914854.
  2. Aluri, J. et al. (2021) Cells 10, 1374. doi: 10.3390/cells10061374.
  3. De Nardo, D. (2015) Cytokine 74, 181-9.
  4. Latty, S.L. et al. (2018) Elife 7:e31377. doi: 10.7554/eLife.31377.
  5. De Nardo, D. et al. (2018) J Biol Chem 293, 15195-15207.
  6. Balka, K.R. and De Nardo, D. (2019) J Leukoc Biol 105, 339-351.
  7. Gold, J.J. et al. (1988) Biosystems 21, 403-15.
  8. Tan, Y. and Kagan, J.C. (2019) Cell 177, 384-398.e11.

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.

    Revision 1
    #39433

    Myddosome Complex Antibody Sampler Kit

    Myddosome Complex Antibody Sampler Kit: Image 1 Expand Image
    Simple Western™ analysis of lysates (0.1 mg/mL) from THP-1 cells using IRAK4 Antibody #4363. 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.
    Myddosome Complex Antibody Sampler Kit: Image 2 Expand Image
    Western blot analysis of serum-starved KARPAS-299 cell extracts, untreated (-) or treated with Human Interleukin-1β (hIL-1β) #8900 (50 ng/ml, 15 min; +), using Phospho-IRAK4 (Thr345/Ser346) (D6D7) Rabbit mAb (upper) or IRAK4 Antibody #4363 (lower). Cell Line Source: Dr Abraham Karpas at the University of Cambridge.
    Myddosome Complex Antibody Sampler Kit: Image 3 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).
    Myddosome Complex Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from Raji, A549, and THP-1 cells using MyD88 (D80F5) Rabbit mAb.
    Myddosome Complex Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from THP-1 (human), RAW 264.7 (mouse), and H-4-II-E (rat) cell lines, using IRAK4 Antibody.
    Myddosome Complex Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from Jurkat, Raji and K562 cell lines, using IRAK2 Antibody #4367.
    Myddosome Complex Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from RD, 293 and A20 cells using IRAK1 (D51G7) Rabbit mAb.
    Myddosome Complex Antibody Sampler Kit: Image 8 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).
    Myddosome Complex Antibody Sampler Kit: Image 9 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.
    Myddosome Complex Antibody Sampler Kit: Image 10 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.
    Myddosome Complex Antibody Sampler Kit: Image 11 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.
    Myddosome Complex Antibody Sampler Kit: Image 12 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.
    Myddosome Complex Antibody Sampler Kit: Image 13 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).
    Myddosome Complex Antibody Sampler Kit: Image 14 Expand Image
    Western blot analysis of extracts from A549 cells, untransfected () or transfected with a MyD88 siRNA (+), using MyD88 (D80F5) Rabbit mAb.
    Myddosome Complex Antibody Sampler Kit: Image 15 Expand Image
    Western blot analysis of extracts from COS-7 cells, either mock transfected or transfected with human IRAK1, using IRAK1 (D51G7) Rabbit mAb.
    Myddosome Complex 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).
    Myddosome Complex Antibody Sampler Kit: Image 17 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.
    Myddosome Complex Antibody Sampler Kit: Image 18 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.
    Myddosome Complex Antibody Sampler Kit: Image 19 Expand Image
    Western blot analysis of extracts from THP-1, mouse embryonic fibroblast (MEF), RAW264.7 and NIH/3T3 cells using IRAK1 (D51G7) Rabbit mAb.
    Myddosome Complex 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).
    Myddosome Complex Antibody Sampler Kit: Image 21 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).
    Myddosome Complex Antibody Sampler Kit: Image 22 Expand Image
    Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® IRAK1 siRNA I (+) or SignalSilence® IRAK1 siRNA II #6228 (+), using IRAK1 (D51G7) Rabbit mAb #4504 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The IRAK1 (D51G7) Rabbit mAb confirms silencing of IRAK1 expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.
    Myddosome Complex Antibody Sampler Kit: Image 23 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.
    Myddosome Complex Antibody Sampler Kit: Image 24 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.
    Myddosome Complex Antibody Sampler Kit: Image 25 Expand Image
    Flow cytometric analysis of untreated Raw264.7 cells, IRAK1 shRNA (blue) or +GFP shRNA (green), using IRAK1 (D51G7) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 fragment (Alexa Fluor® 647 conjugate) #4414 was used as a secondary antibody.
    Myddosome Complex Antibody Sampler Kit: Image 26 Expand Image
    Western blot analysis of extracts from CHO, BHK-21, and A549 cells using MyD88 (D80F5) Rabbit mAb.