Name: vasodilation of isolated vessels and the isolation of the extracellular matrix of tight-skin mice
Uploaded: 2017-03-24T13:00:00
Description: Watch this Scientific Journal Video about Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice at JoVE.com

This work was supported by NIH grants HL-089779 (DW), HL-112270 (KAP) and HL-102836 (KAP) and Cimphoni Life Sciences (part of DW salary). The authors thank Meghann Sytsma for editing the manuscript.

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee (Protocol: AUA#1517). All research involving mice was conducted in conformity with PHS policy.
1. Design of Decoy Peptide
<ol>
	<li>Find the IRF5&#39;s 3D structure and base the design on it. Design a 17 mer, termed IRF5D (ELDWDADDIRLQIDNPD), where aspartate (D) is substit...

<ol>
	<li>Bi, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+interferon+regulatory+factor+5+(IRF5)+expression+in+human+ductal+carcinoma+correlates+with+disease+stage+and+contributes+to+metastasis.">Loss of interferon regulatory factor 5 (IRF5) expression in human ductal carcinoma correlates with disease stage and contributes to metastasis.</a> Breast Cancer Res. 13 (6), 111 (2011).</li><li>Dideberg, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+insertion-deletion+polymorphism+in+the+interferon+regulatory+Factor+5+(IRF5)+gene+confers+risk+of+inflammatory+bowel+diseases.">An insertion-deletion polymorphism in the interferon regulatory Factor 5 (IRF5) gene confers risk of inflammatory bowel diseases.</a> Hum Mol Genet. 16 (24), 3008-3016 (2007).</li><li>Graham, R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+common+haplotype+of+interferon+regulatory+factor+5+(IRF5)+regulates+splicing+and+expression+and+is+associated+with+increased+risk+of+systemic+lupus+erythematosus.">A common haplotype of interferon regulatory factor 5 (IRF5) regulates splicing and expression and is associated with increased risk of systemic lupus erythematosus.</a> Nat.Genet. 38 (5), 550-555 (2006).</li><li>Krausgruber, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IRF5+promotes+inflammatory+macrophage+polarization+and+TH1-TH17+responses.">IRF5 promotes inflammatory macrophage polarization and TH1-TH17 responses.</a> Nat. Immunol. 12 (3), 231-238 (2011).</li><li>Eames, H. L., Corbin, A. L., Udalova, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon+regulatory+factor+5+in+human+autoimmunity+and+murine+models+of+autoimmune+disease.">Interferon regulatory factor 5 in human autoimmunity and murine models of autoimmune disease.</a> Transl Res. 167 (1), 167-182 (2016).</li><li>Mayes, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunochip+analysis+identifies+multiple+susceptibility+Loci+for+systemic+sclerosis.">Immunochip analysis identifies multiple susceptibility Loci for systemic sclerosis.</a> Am J Hum Genet. 94 (1), 47-61 (2014).</li><li>Dimitroulas, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-and+Macrovascular+Treatment+Targets+in+Scleroderma+Heart+Disease.">Micro-and Macrovascular Treatment Targets in Scleroderma Heart Disease.</a> Curr Pharm Des. , (2013).</li><li>Botstein, G. R., LeRoy, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+heart+disease+in+systemic+sclerosis+(scleroderma):+advances+in+clinical+and+pathologic+features,+pathogenesis,+and+new+therapeutic+approaches.">Primary heart disease in systemic sclerosis (scleroderma): advances in clinical and pathologic features, pathogenesis, and new therapeutic approaches.</a> Am Heart J. 102 (5), 913-919 (1981).</li><li>Oram, S., Stokes, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heart+in+scleroderma.">The heart in scleroderma.</a> Br Heart J. 23 (3), 243-259 (1961).</li><li>Xu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4F+decreases+IRF5+expression+and+activation+in+hearts+of+tight-skin+mice.">4F decreases IRF5 expression and activation in hearts of tight-skin mice.</a> PLoS One. 7 (12), 52046 (2012).</li><li>Steen, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heart+in+systemic+sclerosis.">The heart in systemic sclerosis.</a> Curr.Rheumatol.Rep. 6 (2), 137-140 (2004).</li><li>Deshpande, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RCSB+Protein+Data+Bank:+a+redesigned+query+system+and+relational+database+based+on+the+mmCIF+schema.">The RCSB Protein Data Bank: a redesigned query system and relational database based on the mmCIF schema.</a> Nucleic Acids Res. 33, D233-D237 (2005).</li><li>Concepcion, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+detection+of+biomolecular+interactions+using+biolayer+interferometry+for+kinetic+characterization.">Label-free detection of biomolecular interactions using biolayer interferometry for kinetic characterization.</a> Comb Chem High Throughput Screen. 12 (8), 791-800 (2009).</li><li>Matthew, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+biosensor+technologies+in+drug+discovery.">Current biosensor technologies in drug discovery.</a> Drug Discovery. , 69 (2006).</li><li>Doppelt-Azeroual, O., Moriaud, F., Adcock, S. A., Delfaud, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+MED-SuMo+applications.">A review of MED-SuMo applications.</a> Infect Disord Drug Targets. 9 (3), 344-357 (2009).</li><li>Kim, S., Jang, J., Yu, J., Chang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+mucosal+immunization+of+recombinant+adenovirus-based+vaccine+expressing+F1+protein+fragment+induces+protective+mucosal+immunity+against+respiratory+syncytial+virus+infection.">Single mucosal immunization of recombinant adenovirus-based vaccine expressing F1 protein fragment induces protective mucosal immunity against respiratory syncytial virus infection.</a> Vaccine. 28 (22), 3801-3808 (2010).</li><li>Frenzel, D., Willbold, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+Titration+Series+with+Biolayer+Interferometry.">Kinetic Titration Series with Biolayer Interferometry.</a> PloS one. 9 (9), 106882 (2014).</li><li>Ou, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-4F,+an+apolipoprotein+A-1+mimetic,+dramatically+improves+vasodilation+in+hypercholesterolemia+and+sickle+cell+disease.">L-4F, an apolipoprotein A-1 mimetic, dramatically improves vasodilation in hypercholesterolemia and sickle cell disease.</a> Circulation. 107 (18), 2337-2341 (2003).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal.Biochem. 72 (1-2), 248-254 (1976).</li><li>Bauer, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compensatory+phosphorylation+and+protein-protein+interactions+revealed+by+loss+of+function+and+gain+of+function+mutants+of+multiple+serine+phosphorylation+sites+in+endothelial+nitric-oxide+synthase.">Compensatory phosphorylation and protein-protein interactions revealed by loss of function and gain of function mutants of multiple serine phosphorylation sites in endothelial nitric-oxide synthase.</a> J.Biol.Chem. 278 (17), 14841-14849 (2003).</li><li>Weihrauch, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+IRF5+Decoy+Peptide+Reduces+Myocardial+Inflammation+and+Fibrosis+and+Improves+Endothelial+Cell+Function+in+Tight-Skin+Mice.">An IRF5 Decoy Peptide Reduces Myocardial Inflammation and Fibrosis and Improves Endothelial Cell Function in Tight-Skin Mice.</a> PLoS One. 11 (4), 0151999 (2016).</li><li>Hoogenboom, H. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+phage+display+technology+and+its+applications.">Antibody phage display technology and its applications.</a> Immunotechnology. 4 (1), 1-20 (1998).</li><li>Roehm, N. W., Rodgers, G. H., Hatfield, S. M., Glasebrook, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+colorimetric+assay+for+cell+proliferation+and+viability+utilizing+the+tetrazolium+salt+XTT.">An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT.</a> J Immunol Methods. 142 (2), 257-265 (1991).</li><li>Van Tonder, A., Joubert, A. M., Cromarty, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+of+the+3-(4,+5-dimethylthiazol-2-yl)-2,+5-diphenyl-2H-tetrazolium+bromide+(MTT)+assay+when+compared+to+three+commonly+used+cell+enumeration+assays.">Limitations of the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay when compared to three commonly used cell enumeration assays.</a> BMC Res Notes. 8 (1), 1 (2015).</li><li>Ott, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion-decellularized+matrix:+using+nature's+platform+to+engineer+a+bioartificial+heart.">Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart.</a> Nat Med. 14 (2), 213-221 (2008).</li><li>Ou, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-4F,+an+apolipoprotein+A-1+mimetic,+dramatically+improves+vasodilation+in+hypercholesterolemia+and+sickle+cell+disease.">L-4F, an apolipoprotein A-1 mimetic, dramatically improves vasodilation in hypercholesterolemia and sickle cell disease.</a> Circulation. 107 (18), 2337-2341 (2003).</li><li>Weihrauch, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+D-4F+on+vasodilation,+oxidative+stress,+angiostatin,+myocardial+inflammation,+and+angiogenic+potential+in+tight-skin+mice.">Effects of D-4F on vasodilation, oxidative stress, angiostatin, myocardial inflammation, and angiogenic potential in tight-skin mice.</a> Am J Physiol Heart Circ Physiol. 293 (3), 1432-1441 (2007).</li><li>Roy, S., P, P., Mavragani, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IRF-5+-+A+New+Link+to+Autoimmune+Diseases.">IRF-5 - A New Link to Autoimmune Diseases.</a> Autoimmune Disorders - Pathogenetic Aspects. , 35 (2011).</li></ol>

The goal was to design an IRF5 inhibitor to elucidate the role of IRF5 on inflammation, fibrosis, and vascular function in the hearts of Tsk/+ mice. The findings are that IRF5D did not induce proliferation or apoptosis. Moreover, inflammation was reduced and vascular function improved. These data suggest that IRF5 plays an important mechanistic role in the development of inflammation and fibrosis in the heart of Tsk/+ mice and that it has the potential to serve as a therapeutic target.
The fir...

Regulation of cell growth and cell death immune responses is central to the role of the transcription factor family of interferon regulatory factors. IRF5 is highlighted as being crucial for the regulation of immune responses between type 1, an inflammatory promoting response and type 2, an immune response targeting tissue repair. IRF5 is key in cancer1, and autoimmunity2,3,4,5.
The tight-skin mouse (Tsk/+) is a model for tissue fibrosis and scleroderma due to a duplication mutation in the fibrillin-1 gene. This mutation results in a tight-skin and an increase in connective tissue. These mice develop myocardial inflammation, fibrosis and finally heart failure5,6,7,8,9. Scleroderma is an autoimmune fibrotic disorder affecting approximately 150,000 patients in the United States6. The hallmarks of this disease are fibrosis of internal organs including the heart7,8,9,10,11.
The nature of the study demanded the design of an inhibitory peptide. The software approach was chosen over a traditional approach using a phage display. The software approach is easier and less time consuming. The RCSB data bank is used to identify appropriate binding sites12. To study the interaction of the newly designed peptide with the recombinant protein and to focus on the binding parameters, a technique called biolayer interferometry was used. Biolayer interferometry is a biosensor based technique that determines binding affinity, association and disassociation using a biosensor and a binding sample. The biosensor can be fluorescently, luminescently, radiometrically and colorimetrically labeled. The measurement is based on mass addition or depletion resembling association and disassociation13,14. The aim of this study was to understand the role of IRF5 in myocardial inflammation and fibrosis. The goal was to gain insight into the role of IRF5 in the development of tissue fibrosis and scleroderma.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;Triton X 100</td>
			<td>Sigma Aldrich</td>
			<td>X100- 100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 488-labeled goat anti-mouse IgG antibody&#160;</td>
			<td>Thermo Fisher</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bardford reagent</td>
			<td>Thermo Fisher</td>
			<td>23200</td>
			<td>Pierce&#160;</td>
		</tr>
		<tr>
			<td>Biosensors</td>
			<td>Forte-Bio</td>
			<td>MR18-0009</td>
			<td></td>
		</tr>
		<tr>
			<td>CD64 (H-250)</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-15364</td>
			<td></td>
		</tr>
		<tr>
			<td>CellEvent Caspase-3/7 Substrate</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>C10427</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTiter AQueous One Solution Cell Proliferation Assay kit</td>
			<td>Promega</td>
			<td>G3580</td>
			<td>Promega</td>
		</tr>
		<tr>
			<td>DAPI (4&#8242;,6-diamidino-2-phenylindole)</td>
			<td>Thermo Fisher</td>
			<td>D-1306</td>
			<td>1:1000 dilution in PBS</td>
		</tr>
		<tr>
			<td>donkey anti rat Alexa 488</td>
			<td>Thermo Fisher</td>
			<td>A-21208</td>
			<td>1:1000 dilution in PBS</td>
		</tr>
		<tr>
			<td>ECL plus</td>
			<td>GE healthcare/Amersham</td>
			<td>RPN2133</td>
			<td>After a lot of trial and error we came back to this one</td>
		</tr>
		<tr>
			<td>Eclipse TE 200-U microscope with EZ C1 laser scanning software</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti rabbit Alexa 488</td>
			<td>Thermo Fisher</td>
			<td>A-11008</td>
			<td>1:1000 dilution in PBS</td>
		</tr>
		<tr>
			<td>HRP&#160; anti-goat</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-516086</td>
			<td>!:10000 dilution in TBS</td>
		</tr>
		<tr>
			<td>HRP donkey anti-mouse</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-2315</td>
			<td>1:10000 dilution in TBS</td>
		</tr>
		<tr>
			<td>ICAM-1 antibody</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-1511</td>
			<td>1:200 dilution in PBS</td>
		</tr>
		<tr>
			<td>IRF5 antibody (H56)</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-98651</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro plate reader Elx800</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIMP neutrophil marker</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-133821</td>
			<td>1:200 dilution in PBS</td>
		</tr>
		<tr>
			<td>Octet RED</td>
			<td>Forte Bio</td>
			<td></td>
			<td>protein-protein binding</td>
		</tr>
		<tr>
			<td>Peptide design&#160; Medit SA software</td>
			<td>RCSB.org</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant IRF5 protein synthesis</td>
			<td>TopGene Technologies</td>
			<td></td>
			<td>protein expression, synthesis service</td>
		</tr>
		<tr>
			<td>sodium dodecyl phosphate</td>
			<td>Sigma Aldrich</td>
			<td>436143</td>
			<td>detergent</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Pharmacy</td>
			<td></td>
			<td>Schedule III controlled substance, presciption required&#160;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>MedVet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3.5X-45X Trinocular Dissecting Zoom Stereo Microscope with Gooseneck LED Lights</td>
			<td>Am Scope</td>
			<td>SKU: SM-1TSX-L6W</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeba Desalting Columns</td>
			<td>Thermofisher</td>
			<td>2161515</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Basal Media EBM Bullet kit</td>
			<td>Lonza</td>
			<td>CC-3124</td>
			<td>kit contains growth supplemets</td>
		</tr>
		<tr>
			<td>VIA-100K</td>
			<td>&#160;Boeckeler Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4-15% TGX gel</td>
			<td>Bio-Rad</td>
			<td>5671081</td>
			<td></td>
		</tr>
		<tr>
			<td>MedSuMo software</td>
			<td>Medit, Palaiseau, France</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laemmli Buffer</td>
			<td>BioRad</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
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vasodilation of isolated vessels and the isolation of the extracellular matrix of tight-skin mice

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ability to switch cells from one pathway to another is highly attractive as a therapeutic target. We designed a decoy peptide IRF5D with a molecular modeling software for designing small molecules and peptides.
IRF5D inhibited IRF5, reduced alterations in extracellular matrix, and improved endothelial vasodilation in the tight-skin mouse (Tsk/+). The Kd of IRF5D for recombinant IRF5 is 3.72 &plusmn; 0.74 x 10-6 M as determined by binding experiments using biolayer interferometry experiments. Endothelial cells (EC) proliferation and apoptosis were unchanged using increasing concentrations of IRF5D (0 to 100 &#181;g/mL, 24 h). Tsk/+ mice were treated with IRF5D (1 mg/kg/d subcutaneously, 21 d). IRF5 and ICAM expressions were decreased after IRF5D treatment. Endothelial function was improved as assessed by vasodilation of facialis arteries from Tsk/+ mice treated with IRF5D compared to Tsk/+ mice without IRF5D treatment. As a transcription factor, IRF5 traffics from the cytosol to the nucleus. Translocation was assessed by immunohistochemistry on cardiac myocytes cultured on the different cardiac extracellular matrices. IRF5D treatment of the Tsk/+ mouse resulted in a reduced number of IRF5 positive nuclei in comparison to the animals without IRF5D treatment (50 &#181;g/mL, 24 h). These findings demonstrate the important role that IRF5 plays in inflammation and fibrosis in Tsk/+ mice.

The results demonstrated in Figure 1 show how to design a peptide. Figure 1, upper left, shows the region (between the 2 yellow arrows, amino acids (aa) 425-436) in IRF5 that is phosphorylated by a number of kinases. Figure 1, upper right, shows a yellow oval where IRF5&#39;s phosphorylated domain binds. The dimeric structure of 3DSH was rotated to observe a cleft or valley to the left of the Helix 2 (aa303-312). This is where the phospho...

Watch this Scientific Journal Video about Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice at JoVE.com

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice

We describe the isolation of cardiac extracellular matrix from C57Bl/6J control mice, tight-skin mice, and tight-skin mice treated with the IRF5 inhibitory peptide. We also describe the vasodilation studies on the isolated vessels from C57Bl/6J, tight-skin mice and tight-skin mice treated with the IRF5 inhibitory peptide.

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ...

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Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Isolation and Th17 Differentiation of NaÃƒÆ’Ã‚Â¯ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...