Preparation of 5 mM VIPER and CP7 Stock SolutionsNote: 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.

VIPER: A TLR4 Inhibitory Peptide: 1 mg (lyophilized) KYSFKLILAEYRRRRRRRRR (VIPER sequence: KYSFKLILAEY). Molecular weight: 2780.3CP7, Control Peptide: 1 mg (lyophilized) RNTISGNIYSARRRRRRRRR (Control sequence: RNTISGNIYSA). Molecular weight: 2601

TLR4

>95%, by HPLC.

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. Use in Function reported in scientific literature (PMID: 24630524). Use in blocking / neutralizing reported in multiple pieces of scientific literatureFLOW application reported by Alvarez et al - Conference on Retroviruses and Opportunistic Infections - CROI 2014 (Abstract: The NCOR2-Nurr1-CoREST Transrepression Axis Impairs HIV Reactivation in Latently Infected Microglial Cells)Use in Binding inhibition reported in scientific literature (PMID: 27897392).