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TLR4 Inhibitor Peptide Set

Images

 
TLR4 Inhibitor Peptide Set [NBP2-26244] - The inhibitor peptide inhibits TLR4 signaling by blocking interactions between TLR4 and its adaptors Mal/TIRAP and TRAM.
Blocking/Neutralizing: TLR4 Inhibitor Peptide Set [NBP2-26244] - iBMDM cultures were pretreated with 5uM of inhibitor or control prior to stimulation with 20 ng/ml LPS. Negative (LPS-, LPS-/CP7+, LPS-/CP7+) and positive ...read more
TLR4 Inhibitor Peptide Set [NBP2-26244] - TLR4/MD-2/CD14/NF-kB/SEAPorter stably transfected cells were plated in 96-well plates at 1x10^5 cells/well. After 16 h, cells were preincubated with various concentrations of ...read more

Product Details

Summary
Reactivity Hu, Mu, RtSpecies Glossary
Applications Flow, Func, B/N
Concentration
LYOPH

Order Details

TLR4 Inhibitor Peptide Set Summary

Description
TLR4 inhibitor and control peptides are quality controlled in vitro using the TLR4/MD-2/CD14/NF-kB/SEAP cell line with SEAP as a readout assay (Image 3).
Preparation
Method
Preparation of 5 mM VIPER and CP7 Stock Solutions
Note: Bring the peptides to room temperature and quick spin the tubes before opening the caps.

VIPER: A final volume of 72 ul will make a 5 mM stock solution. Add 72 ul sterile H20 to the tube of peptide. Carefully pipet to ensure all of the peptide is dissolved.

CP7: A final volume of 76 ul will make a 5 mM stock solution. Add 76 ul sterile H20 to the tube of peptide. Carefully pipet to ensure all of the peptide is dissolved.

The stock solutions may be diluted further to make working solutions. Dilute according to the needs for your assay. For example dilute 5 mM stock solutions 1:10 in sterile 1X PBS or cell culture media to make 500 uM working solutions. Working solutions should be made fresh daily and not stored.
Content
VIPER: A TLR4 Inhibitory Peptide: 1 mg (lyophilized) KYSFKLILAEYRRRRRRRRR (VIPER sequence: KYSFKLILAEY). Molecular weight: 2780.3

CP7, Control Peptide: 1 mg (lyophilized) RNTISGNIYSARRRRRRRRR (Control sequence: RNTISGNIYSA). Molecular weight: 2601
Gene
TLR4
Purity
>95%, by HPLC.

Applications/Dilutions

Dilutions
  • Binding Inhibition reported in scientific literature (PMID 27897392)
  • Block/Neutralize reported in multiple pieces of scientific literature
  • Flow Cytometry
  • Functional reported in scientific literature (PMID 24630524)
  • In vitro assay
Application Notes
The inhibitor is used in assays to inhibit TLR4 activation; see Image 3 and also refer to Lysakova-Devine et al (2010) for examples. Optimal inhibitor concentrations should be established through titration and may vary between model systems. We recommend an initial titration from 0-30 uM for in vitro assays (Image 3). Control concentrations should mirror inhibitor concentrations. Inhibitor and control should be preincubated with cells prior to ligand activation to allow sufficient time for the peptides to enter from the media into the cell. We typically preincubate with inhibitor and control peptides for 2 h prior to TLR4 activation with LPS (Image 3); however, optimal preincubation times may vary between model systems.

The TLR4/MD-2/CD14 stably transfected cell line is a useful positive control model system for studying inhibition of TLR4 activation by VIPER (Image 3). SEAP is used as a readout assay in Image 3 to measure TLR4 inhibition.

A novel model system is shown in Image 1 where TLR4 inhibitor peptide, but not CP7, inhibited TLR4 activation in Mal-deficient immortalized mouse bone marrow-derived macrophages (iBMDMs). In these iBMDMs, the inhibitor targets TLR4-TRAM, but not TLR-Mal, interactions as Mal is not expressed. TNF-alpha is used as a readout assay in Image 1 to measure inhibition.
Reviewed Applications
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NBP2-26244 in the following applications:

Publications
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NBP2-26244 in the following applications:

Reactivity Notes

Rat reactivity reported in scientific literature (PMID: 25811788)

Packaging, Storage & Formulations

Storage
Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.
Concentration
LYOPH
Purity
>95%, by HPLC.
Reconstitution Instructions
Please contact technical support for detailed reconstitution instructions.

Alternate Names for TLR4 Inhibitor Peptide Set

  • ARMD10
  • CD_antigen: CD284
  • CD284 antigen
  • CD284
  • EC 3.2.2.6
  • EC:3.2.2.6
  • homolog of Drosophila toll
  • hToll
  • TLR4
  • TLR-4
  • toll like receptor 4 protein
  • TOLL
  • toll-like receptor 4

Background

TLR4 (Toll-like receptor 4) is a type-1 transmembrane glycoprotein that is a pattern recognition receptor (PRR) belonging to the TLR family (1-3). TLR4 is expressed in many tissues and is most abundantly expressed in the placenta, spleen, and peripheral blood leukocytes (1). Human TLR4 is synthesized as a 839 amino acid (aa) protein containing a signal sequence (1-23 aa), an extracellular domain (ECD) (24-631 aa), a transmembrane domain (632-652 aa), and Toll/interleukin-1 receptor (TIR) cytoplasmic domain (652-839 aa) with a theoretical molecular weight of 95 kDa (3, 4). The ECD contains 21 leucine-rich repeats (LRRs) and has a horseshoe-shaped structure (3, 4). TLR4 requires binding with the co-receptor myeloid differentiation protein 2 (MD2) largely via hydrophilic interactions for proper ligand sensing and signaling (2-4). In general, the TLR family plays a role in activation of innate immunity and responds to a variety of pathogen-associated molecular patterns (PAMPs) (5). TLR4 is specifically responsive to lipopolysaccharide (LPS), which is found on the outer-membrane of most ram-negative bacteria (3-5). Activation of TLR4 requires binding of a ligand, such as LPS to MD2, followed by MD2-LPS complex binding to TLR4, resulting in a partial complex (TLR4-MD2/LPS) (3, 5). To become fully active, two partial complexes must dimerize thereby allowing the TIR domains of TLR4 to bind other adapter molecular and initiate signaling, triggering an inflammatory response and cytokine production (3, 5).

TLR4 signaling occurs through two distinct pathways: The MyD88 (myeloid differentiation primary response gene 88)-dependent pathway and the MyD88-independent (TRIF-dependent, TIR domain-containing adaptor inducing IFN-beta) pathway (3, 5-7). The MyD88-dependent pathway occurs mainly at the plasma membrane and involves the binding of MyD88-adaptor-like (MAL) protein followed by a signaling cascade that results in the activation of transcription factors including nuclear factor-kappaB (NF-kappaB) that promote the secretion of inflammatory molecules and increased phagocytosis (5-7). Conversely, the MyD88-independent pathway occurs after TLR4-MD2 complex internalization in the endosomal compartment. This pathway involves the binding of adapter proteins TRIF and TRIF-related adaptor molecule (TRAM), a signaling activation cascade resulting in IFN regulatory factor 3 (IRF3) translocation into the nucleus, and secretion of interferon-beta (INF-beta) genes and increased phagocytosis (5-7).

Given its expression on immune-related cells and its role in inflammation, TLR4 activation can contribute to various diseases (6-8). For instance, several studies have found that TLR4 activation is associated with neurodegeneration and several central nervous system (CNS) pathologies, including Alzheimer's disease, Parkinson's disease, and Huntington's disease (6, 7). Furthermore, TLR4 mutations have been shown to lead to higher rates of infections and increased susceptibility to sepsis (7-8). One potential therapeutic approach aimed at targeting TLR4 and neuroinflammation is polyphenolic compounds which include flavonoids and phenolic acids and alcohols (8).

Alternative names for TLR4 includes 76B357.1, ARMD10, CD284 antigen, CD284, EC 3.2.2.6, homolog of Drosophila toll, hToll, toll like receptor 4 protein, TOLL, toll-like receptor 4.

References

1. Vaure, C., & Liu, Y. (2014). A comparative review of toll-like receptor 4 expression and functionality in different animal species. Frontiers in immunology. https://doi.org/10.3389/fimmu.2014.00316

2. Park, B. S., & Lee, J. O. (2013). Recognition of lipopolysaccharide pattern by TLR4 complexes. Experimental & molecular medicine. https://doi.org/10.1038/emm.2013.97

3. Krishnan, J., Anwar, M.A., & Choi, S. (2016) TLR4 (Toll-Like Receptor 4). In: Choi S. (eds) Encyclopedia of Signaling Molecules. Springer, New York, NY. https://doi.org/10.1007/978-1-4614-6438-9_592-1

4. Botos, I., Segal, D. M., & Davies, D. R. (2011). The structural biology of Toll-like receptors. Structure. https://doi.org/10.1016/j.str.2011.02.004

5. Lu, Y. C., Yeh, W. C., & Ohashi, P. S. (2008). LPS/TLR4 signal transduction pathway. Cytokine. https://doi.org/10.1016/j.cyto.2008.01.006

6. Leitner, G. R., Wenzel, T. J., Marshall, N., Gates, E. J., & Klegeris, A. (2019). Targeting toll-like receptor 4 to modulate neuroinflammation in central nervous system disorders. Expert opinion on therapeutic targets. https://doi.org/10.1080/14728222.2019.1676416

7. Molteni, M., Gemma, S., & Rossetti, C. (2016). The Role of Toll-Like Receptor 4 in Infectious and Noninfectious Inflammation. Mediators of inflammation. https://doi.org/10.1155/2016/6978936

8. Rahimifard, M., Maqbool, F., Moeini-Nodeh, S., Niaz, K., Abdollahi, M., Braidy, N., Nabavi, S. M., & Nabavi, S. F. (2017). Targeting the TLR4 signaling pathway by polyphenols: A novel therapeutic strategy for neuroinflammation. Ageing research reviews. https://doi.org/10.1016/j.arr.2017.02.004

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Inhibitors are guaranteed for 1 year from date of receipt.

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Publications for TLR4 Inhibitor (NBP2-26244)(47)

We have publications tested in 3 confirmed species: Human, Mouse, Rat.

We have publications tested in 9 applications: B/N, BindInhib, FLOW, Forced Spectroscopy, Func, In Vivo, In vitro, In-vitro, In-vivo.


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(11)
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(13)
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(1)
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(9)
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(1)
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Showing Publications 1 - 10 of 47. Show All 47 Publications.
Publications using NBP2-26244 Applications Species
Verma S, Reddy P, Sowdhamini R Integrated approaches for the recognition of small molecule inhibitors for Toll-like receptor 4 Computational and Structural Biotechnology Journal 2023-07-22 [PMID: 37576745] (In vitro) In vitro
Yang D, Dai X, Xing Y et al. Intrinsic cardiac adrenergic cells contribute to LPS-induced myocardial dysfunction Communications biology 2022-01-25 [PMID: 35079095]
Yamamoto K, Kondo Y, Ohnishi S Et al. The TLR4-TRIF-type 1 IFN-IFN- gamma pathway is crucial for gastric MALT lymphoma formation after Helicobacter suis infection iScience 2021-08-01 [PMID: 34585114] (In vitro) In vitro
Gergen AK, Jarrett MJ, Li A et al. Toll-like Receptor 4 Mediates Reflux-Induced Inflammation in a Murine Reflux Model Seminars in thoracic and cardiovascular surgery 2021-09-14 [PMID: 34534678]
Mizuno Y, Taguchi T Self-assembled dodecyl group-modified gelatin microparticle-based hydrogels with angiogenic properties NPG Asia Mater 2020-12-01
Li B, Yan C, Wu J et al. Clonorchis sinensis ESPs enhance the activation of hepatic stellate cells by a cross-talk of TLR4 and TGF-beta/Smads Signaling pathway Acta Trop. 2019-12-17 [PMID: 31862462] (Func) Func
Harada Y, Zhang J, Imari K et al. Cathepsin E in neutrophils contributes to the generation of neuropathic pain in experimental autoimmune encephalomyelitis Pain 2019-09-01 [PMID: 31095099] (Mouse) Mouse
Chehimi M, Ward R, Pestel J et al. Omega-3 Polyunsaturated Fatty Acids Inhibit IL-17A Secretion through Decreased ICAM-1 Expression in T Cells Co-Cultured with Adipose-Derived Stem Cells Harvested from Adipose Tissues of Obese Subjects Mol Nutr Food Res 2019-03-08 [PMID: 30848861] (B/N, Human) B/N Human
Tun X, Yasukawa K, Yamada KI. Nitric Oxide Is Involved in Activation of Toll-Like Receptor 4 Signaling through Tyrosine Nitration of Src Homology Protein Tyrosine Phosphatase 2 in Murine Dextran Sulfate-Induced Colitis Biol. Pharm. Bull. 2018-12-04 [PMID: 30504685] (B/N, Mouse) B/N Mouse
Nishida M, Saegusa J, Tanaka S, Morinobu A. S100A12 facilitates osteoclast differentiation from human monocytes. PLoS ONE 2018-09-20 [PMID: 30235276] (Human) Human
Show All 47 Publications.

Review for TLR4 Inhibitor (NBP2-26244) (1) 51

Average Rating: 5
(Based on 1 review)
We have 1 review tested in 1 species: Human.

Reviews using NBP2-26244:
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ELISA TLR4 NBP2-26244
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5
reviewed by:
anonymous aa
ELISA Human 09/15/2014
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Summary

ApplicationELISA
Sample TestedTHP-1 cell culture supernatant
SpeciesHuman

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PAMPs and DAMPs: What is the Same and What is Different About These Molecules?
By Victoria OsinskiWhat are PAMPs and DAMPsInflammation results from stimuli signaling damage or infection. The immune system inflammatory response can be beneficial or harmful depending on the type and duration of ...  Read full blog post.

How To Identify B Cell Subsets Using Flow Cytometry
By Victoria OsinskiUsing Flow Cytometry to Identify B Cell SubsetsIdentifying cellular subsets by flow cytometry requires careful and thorough planning in order to ensure the correct subset of cells are identified...  Read full blog post.

Lipopolysaccharide from gut microbiome localizes in human atherosclerotic plaques and promotes TLR4-mediated oxidative stress
By Jamshed Arslan, Pharm. D., PhD. Atherosclerosis is a chronic inflammatory condition in which plaques of fats and other substances slowly buildup on the inner walls of arteries to restrict blood flow. In atheroscle...  Read full blog post.

Toll-like receptors in the intestinal epithelial cells
By Jamshed Arslan, Pharm. D., PhD. Toll-like receptors (TLRs) are microbe-sensing proteins that act as first responders to danger signals. TLRs help the intestinal epithelial cells (IECs) recognize commensal bacteria ...  Read full blog post.

The role of STING/TMEM173 in gamma and encephalitis Herpes Simplex Virus (HSV)
Stimulator of interferon genes (STING), also known as TMEM173, promotes the production of the interferon’s IFN-alpha and IFN-beta.  STING possesses three functional domains: a cytoplasmic C-terminal tail, a central globular domain, and four N-...  Read full blog post.

TRIF/TICAM1 and mitochondrial dynamics in the innate immune response
TRIF, also known as toll like receptor adaptor molecule 1 or TICAM1, is known for its role in invading foreign pathogens as part of our innate immune response. TRIF/TICAM1 is a TIR-domain adaptor protein (toll/interleukin-1 receptor) that interacts...  Read full blog post.

The role of TLR4 in breast cancer
Toll like receptors (TLRs) are highly conserved proteins that are first known for their role in pathogen recognition and immune response activation.  In order to elicit the necessary immune response in reaction to a foreign pathogen, TLRs trigger cy...  Read full blog post.

cIAP2 - balancing cell death and cell survival
The inhibitor of apoptosis proteins (IAPs) are important regulators of cell death and inflammation. The cellular inhibitor of apoptosis protein 2 (cIAP2) contains three Baculovirus IAP repeat (BIR) domains, a Ubiquitin associated (UBA) domain, and ...  Read full blog post.

TLR4 - A Guardian of Innate Immunity
Toll-like receptor 4 (TLR4) belongs to the family of Toll-like receptors (TLR), and plays a main role in pathogen recognition and innate immunity system activation. The TLR family members are highly conserved proteins that all contain a high degree of...  Read full blog post.

IRAK4: The "master IRAK" critical for initiating immune responses
IRAK4, also known as Interleukin-1 receptor-associated kinase 4, is a serine/threonine-protein kinase that plays a critical role in initiating innate and adaptive immune responses against foreign pathogens. It activates NF-kappaB in both Toll-like rec...  Read full blog post.

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anonymous aa
09/15/2014
Application: ELISA
Species: Human

Bioinformatics

Gene Symbol TLR4