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39433
Myddosome Complex Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Myddosome Complex Antibody Sampler Kit #39433

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Myddosome Complex Antibody Sampler Kit: Image 1
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 2
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 3
Western blot analysis of extracts from Raji, A549, and THP-1 cells using MyD88 (D80F5) Rabbit mAb.
Myddosome Complex Antibody Sampler Kit: Image 4
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 5
Western blot analysis of extracts from Jurkat, Raji and K562 cell lines, using IRAK2 Antibody #4367.
Myddosome Complex Antibody Sampler Kit: Image 6
Western blot analysis of extracts from RD, 293 and A20 cells using IRAK1 (D51G7) Rabbit mAb.
Myddosome Complex Antibody Sampler Kit: Image 7
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 8
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 9
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 10
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 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.
Myddosome Complex Antibody Sampler Kit: Image 12
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 13
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 14
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 15
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 16
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 17
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 18
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 19
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 20
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 21
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 22
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 23
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.
To Purchase # 39433
Cat. # Size Qty. Price
39433T
1 Kit  (6 x 20 microliters) $ 592

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
MyD88 (D80F5) Rabbit mAb 4283 20 µl
  • WB
  • IP
H M R Mk 33 Rabbit IgG
IRAK1 (D51G7) Rabbit mAb 4504 20 µl
  • WB
  • IP
  • F
H M Mk 78-105 Rabbit IgG
IRAK2 Antibody 4367 20 µl
  • WB
H M R Mk 62 Rabbit 
IRAK4 Antibody 4363 20 µl
  • WB
  • IP
H M R Mk 55 Rabbit 
Phospho-IRAK4 (Thr345/Ser346) (D6D7) Rabbit mAb 11927 20 µl
  • WB
  • IP
H 55 Rabbit IgG
TRAF6 (E2K9D) Rabbit mAb 67591 20 µl
  • WB
  • IP
H M R 60 Rabbit IgG
TBK1/NAK (E8I3G) Rabbit mAb 38066 20 µl
  • WB
  • IP
  • IF
  • F
H M R 84 Rabbit IgG
Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb 5483 20 µl
  • WB
  • IP
  • IF
  • F
H M 84 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product 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.

Specificity / Sensitivity

Each antibody in the Myddosome Complex Antibody Sampler Kit detects endogenous levels of its target human protein. Phospho-IRAK4 (Thr345/Ser346) (D6D7) Rabbit mAb recognizes endogenous levels of IRAK4 protein when phosphorylated at Thr345 and Ser346. This antibody shows slight reactivity with IRAK4 when singly phosphorylated at Ser346 and does not cross-react with IRAK4 singly phosphorylated at Thr345. Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb detects endogenous levels of TBK1 only when phosphorylated at Ser172 and may cross-react with phospho-IKKε.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with recombinant protein specific to a central region of mouse TRAF6 proteins, with synthetic phosphopeptides corresponding to residues surrounding Thr345/Ser346 and Ser172 of human IRAK4 and TBK1/NAK1 proteins, respectively, and with synthetic peptides corresponding to residues surrounding Cys233 of human MyD88 protein and residues near the carboxyl termini of mouse IRAK1 and human TBK1/NAK protein. Polyclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues at the carboxy terminus of mouse IRAK2 and residues surrounding Lys41 of human IRAK4. Antibodies were purified by protein A and peptide affinity chromatography.

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.

Pathways & Proteins

Explore pathways + proteins related to this product.

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.

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