Revision 1

#39433

Store at -20C

Myddosome Complex Antibody Sampler Kit

1 Kit

(6 x 20 microliters)

CST Logo
Orders:

877-616-CELL (2355)

[email protected]

Support:

877-678-TECH (8324)

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

For Research Use Only. Not for Use in Diagnostic Procedures.

Product Includes Product # Quantity Mol. Wt Isotype/Source
MyD88 (D80F5) Rabbit Monoclonal Antibody428320 µl33 kDaRabbit IgG
IRAK1 (D51G7) Rabbit Monoclonal Antibody450420 µl78-105 kDaRabbit IgG
IRAK2 Antibody436720 µl62 kDaRabbit
IRAK4 Antibody436320 µl55 kDaRabbit
Phospho-IRAK4 (Thr345/Ser346) (D6D7) Rabbit Monoclonal Antibody1192720 µl55 kDaRabbit IgG
TRAF6 (E2K9D) Rabbit Monoclonal Antibody6759120 µl60 kDaRabbit IgG
TBK1/NAK (E8I3G) Rabbit Monoclonal Antibody3806620 µl84 kDaRabbit IgG
Phospho-TBK1/NAK (Ser172) (D52C2) Rabbit Monoclonal Antibody548320 µl84 kDaRabbit IgG
Anti-rabbit IgG, HRP-linked Antibody7074100 µlGoat

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).

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

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

Cell Signaling Technology Logo
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.
undefined Image 1: Myddosome Complex Antibody Sampler Kit
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).
undefined Image 2: Myddosome Complex Antibody Sampler Kit
Western blot analysis of extracts from Raji, A549, and THP-1 cells using MyD88 (D80F5) Rabbit mAb.
undefined Image 3: Myddosome Complex Antibody Sampler Kit
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

Cell Signaling Technology Logo
Western blot analysis of extracts from THP-1 (human), RAW 264.7 (mouse), and H-4-II-E (rat) cell lines, using IRAK4 Antibody.
undefined Image 4: Myddosome Complex Antibody Sampler Kit
Western blot analysis of extracts from Jurkat, Raji and K562 cell lines, using IRAK2 Antibody #4367.
undefined Image 5: Myddosome Complex Antibody Sampler Kit
Western blot analysis of extracts from RD, 293 and A20 cells using IRAK1 (D51G7) Rabbit mAb.
undefined Image 6: Myddosome Complex Antibody Sampler Kit
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

Cell Signaling Technology Logo
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).
undefined Image 7: Myddosome Complex Antibody Sampler Kit
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.
undefined Image 8: Myddosome Complex Antibody Sampler Kit
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.
undefined Image 9: Myddosome Complex Antibody Sampler Kit
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

Cell Signaling Technology Logo
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.
undefined Image 10: Myddosome Complex Antibody Sampler Kit
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.
undefined Image 11: Myddosome Complex Antibody Sampler Kit
Western blot analysis of extracts from various cell lines using TBK1/NAK (E8I3G) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
undefined Image 12: Myddosome Complex Antibody Sampler Kit
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

Cell Signaling Technology Logo
Western blot analysis of extracts from A549 cells, untransfected () or transfected with a MyD88 siRNA (+), using MyD88 (D80F5) Rabbit mAb.
undefined Image 13: Myddosome Complex Antibody Sampler Kit
Western blot analysis of extracts from COS-7 cells, either mock transfected or transfected with human IRAK1, using IRAK1 (D51G7) Rabbit mAb.
undefined Image 14: Myddosome Complex Antibody Sampler Kit
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).
undefined Image 15: Myddosome Complex Antibody Sampler Kit
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

Cell Signaling Technology Logo
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.
undefined Image 16: Myddosome Complex Antibody Sampler Kit
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.
undefined Image 17: Myddosome Complex Antibody Sampler Kit
Western blot analysis of extracts from THP-1, mouse embryonic fibroblast (MEF), RAW264.7 and NIH/3T3 cells using IRAK1 (D51G7) Rabbit mAb.
undefined Image 18: Myddosome Complex Antibody Sampler Kit
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

Cell Signaling Technology Logo
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).
undefined Image 19: Myddosome Complex Antibody Sampler Kit
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).
undefined Image 20: Myddosome Complex Antibody Sampler Kit
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.
undefined Image 21: Myddosome Complex Antibody Sampler Kit
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

Cell Signaling Technology Logo
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.
undefined Image 22: Myddosome Complex Antibody Sampler Kit
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.
undefined Image 23: Myddosome Complex Antibody Sampler Kit
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.
undefined Image 24: Myddosome Complex Antibody Sampler Kit
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

Cell Signaling Technology Logo
Western blot analysis of extracts from CHO, BHK-21, and A549 cells using MyD88 (D80F5) Rabbit mAb.
undefined Image 25: Myddosome Complex Antibody Sampler Kit
Simple Western™ analysis of lysates (1 mg/mL) from Raji cells using MyD88 (D80F5) Rabbit mAb #4283. 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.
undefined Image 26: Myddosome Complex Antibody Sampler Kit
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.
undefined Image 27: Myddosome Complex Antibody Sampler Kit
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

Cell Signaling Technology Logo
Western blot analysis of extracts from THP-1 cells, undifferentiated (-) or differentiated with TPA (80 nM, overnight) followed by treatment with LPS (1 µg/ml, 8 hrs; +), using TBK1/NAK (E9H5S) Mouse mAb #51872 (Panel A) and Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb #5483 (Panel B). Anti-mouse IgG (H+L) (DyLight 680 Conjugate) #5470 (red) and Anti-rabbit IgG (H+L) (DyLight 800 4X PEG Conjugate) #5151 (green) were used as secondary antibodies.
undefined Image 28: Myddosome Complex Antibody Sampler Kit
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.