TLR4 Inhibitor
Novus Biologicals, part of Bio-Techne | Catalog # NBP2-31230
Key Product Details
Species
Human, Mouse
Applications
Functional Assay
Product Specifications
Specificity
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 (Fig 3).
Application Notes
The inhibitor is used in assays to inhibit TLR4 activation; see 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. 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 (Fig 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 (Fig 3). SEAP is used as a readout assay in Figure 3 to measure TLR4 inhibition.
A novel model system is shown in Figure 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 Figure 1 to measure inhibition.
The TLR4/MD-2/CD14 stably transfected cell line is a useful positive control model system for studying inhibition of TLR4 activation by VIPER (Fig 3). SEAP is used as a readout assay in Figure 3 to measure TLR4 inhibition.
A novel model system is shown in Figure 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 Figure 1 to measure inhibition.
Inhibitor Content
VIPER: A TLR4 Inhibitory Peptide: 1 mg (lyophilized) KYSFKLILAEYRRRRRRRRR (VIPER sequence: KYSFKLILAEY). Molecular weight: 2780.3
Formulation, Preparation, and Storage
Preparation Method
Preparation of 5 mM VIPER 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.
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.
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.
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.
Formulation
Lyophilized white powder
Concentration
Lyoph
Reconstitution Instructions
Please contact technical support for detailed reconstitution instructions.
Shipping
The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Storage
Store at -20C. Avoid freeze-thaw cycles.
Background: TLR4
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
Long Name
Toll-like Receptor 4
Alternate Names
CD284
Gene Symbol
TLR4
Additional TLR4 Products
Product Specific Notices for TLR4 Inhibitor
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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