Name: in vitro analysis of pdz-dependent cftr macromolecular signaling complexes
Uploaded: 2012-08-13T13:00:00
Description: Watch this Scientific Journal Video about In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes at JoVE.com

Çalışmalarımız Amerikan Kalp Derneği (Büyük Güneydoğu Ortaklık) Başlangıç-devlet yardımı 0765185B, Elsa U. Pardee Vakfı&#39;nın araştırma bursu ve Wayne State Üniversitesi intramural başlangıç ​​fonu ve Damar Araştırma Enstitüsü İsis Girişimi ödülü gelen hibelerle desteklenmektedir. In vitro CFTR makromoleküler karmaşık montaj Bu yöntem başlangıçta Dr AP Naren (Tennessee Sağlık Bilimleri Merkezi Üniversitesi) tarafından öncülük edilmiştir.

1. Bakterilerde Rekombinant Takip edilen Fusion Proteinlerin İfade ve Arıtma <ol><li> CFTR, LPA 2, MRP2, MRP4, β 2 AR ve NHERFs (tam boy veya PDZ1 ya PDZ2 etki için C-kuyrukları (C-terminusta PDZ motifleri ihtiva eden son 50-100 amino asitleri) ile tanımlanan bölgeler amplifiye ) PCR yaklaşım. </li><li> GST-füzyon proteinleri için pGEX4T-1 vektör (örneğin GST-NHERFs, GST-MRP4 CT), MBP-füzyon proteinleri için pMAL-C2 vektör (örneğin MBP-β 2 AR CT, MBP-CFTR CT içine ...

<ol>
	<li>Anderson, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+that+CFTR+is+a+chloride+channel+by+alteration+of+its+anion+selectivity.">Demonstration that CFTR is a chloride channel by alteration of its anion selectivity.</a> Science. 253, 202-205 (1991).</li><li>Bear, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+functional+reconstitution+of+the+cystic+fibrosis+transmembrane+conductance+regulator+(CFTR.">Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR.</a> Cell. 68, 809-818 (1992).</li><li>Quinton, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloride+impermeability+in+cystic+fibrosis.">Chloride impermeability in cystic fibrosis.</a> Nature. 301, 421-422 (1983).</li><li>Cheng, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+intracellular+transport+and+processing+of+CFTR+is+the+molecular+basis+of+most+cystic+fibrosis.">Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis.</a> Cell. 63, 827-834 (1990).</li><li>Gabriel, S. E., Brigman, K. N., Koller, B. H., Boucher, R. C., Stutts, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis+heterozygote+resistance+to+cholera+toxin+in+the+cystic+fibrosis+mouse+model.">Cystic fibrosis heterozygote resistance to cholera toxin in the cystic fibrosis mouse model.</a> Science. 266, 107-109 (1994).</li><li>Li, C., Naren, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CFTR+chloride+channel+in+the+apical+compartments:+spatiotemporal+coupling+to+its+interacting+partners.">CFTR chloride channel in the apical compartments: spatiotemporal coupling to its interacting partners.</a> Integr. Biol (Camb). 2, 161-177 (2010).</li><li>Chao, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+intestinal+CFTR+Cl-+channel+by+heat-stable+enterotoxin+and+guanylin+via+cAMP-dependent+protein+kinase.">Activation of intestinal CFTR Cl- channel by heat-stable enterotoxin and guanylin via cAMP-dependent protein kinase.</a> Embo. J. 13, 1065-1072 (1994).</li><li>Gabriel, S. E., Clarke, L. L., Boucher, R. C., Stutts, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CFTR+and+outward+rectifying+chloride+channels+are+distinct+proteins+with+a+regulatory+relationship.">CFTR and outward rectifying chloride channels are distinct proteins with a regulatory relationship.</a> Nature. 363, 263-268 (1993).</li><li>McNicholas, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+of+a+renal+K++channel+(ROMK2)+to+the+inhibitory+sulfonylurea+compound+glibenclamide+is+enhanced+by+coexpression+with+the+ATP-binding+cassette+transporter+cystic+fibrosis+transmembrane+regulator.">Sensitivity of a renal K+ channel (ROMK2) to the inhibitory sulfonylurea compound glibenclamide is enhanced by coexpression with the ATP-binding cassette transporter cystic fibrosis transmembrane regulator.</a> Proc. Natl. Acad. Sci. USA. 93, 8083-8088 (1996).</li><li>Schreiber, R., Nitschke, R., Greger, R., Kunzelmann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cystic+fibrosis+transmembrane+conductance+regulator+activates+aquaporin+3+in+airway+epithelial+cells.">The cystic fibrosis transmembrane conductance regulator activates aquaporin 3 in airway epithelial cells.</a> J. Biol. Chem. 274, 11811-11816 (1999).</li><li>Shumaker, H., Amlal, H., Frizzell, R., Ulrich, C. D., Soleimani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CFTR+drives+Na+-nHCO-3+cotransport+in+pancreatic+duct+cells:+a+basis+for+defective+HCO-3+secretion+in+CF.">CFTR drives Na+-nHCO-3 cotransport in pancreatic duct cells: a basis for defective HCO-3 secretion in CF.</a> Am. J. Physiol. 276, 16-25 (1999).</li><li>Ahn, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+interaction+between+the+cystic+fibrosis+transmembrane+conductance+regulator+and+HCO3-+salvage+mechanisms+in+model+systems+and+the+mouse+pancreatic+duct.">Regulatory interaction between the cystic fibrosis transmembrane conductance regulator and HCO3- salvage mechanisms in model systems and the mouse pancreatic duct.</a> J. Biol. Chem. 276, 17236-17243 (2001).</li><li>Sugita, M., Yue, Y., Foskett, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CFTR+Cl-+channel+and+CFTR-associated+ATP+channel:+distinct+pores+regulated+by+common+gates.">CFTR Cl- channel and CFTR-associated ATP channel: distinct pores regulated by common gates.</a> Embo. J. 17, 898-908 (1998).</li><li>Naren, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+CFTR+chloride+channels+by+syntaxin+and+Munc18+isoforms.">Regulation of CFTR chloride channels by syntaxin and Munc18 isoforms.</a> Nature. 390, 302-305 (1997).</li><li>Naren, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syntaxin+1A+is+expressed+in+airway+epithelial+cells,+where+it+modulates+CFTR+Cl(-)+currents.">Syntaxin 1A is expressed in airway epithelial cells, where it modulates CFTR Cl(-) currents.</a> J. Clin. Invest. 105, 377-386 (2000).</li><li>Naren, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+macromolecular+complex+of+beta+2+adrenergic+receptor,+CFTR,+and+ezrin/radixin/moesin-binding+phosphoprotein+50+is+regulated+by+PKA.">A macromolecular complex of beta 2 adrenergic receptor, CFTR, and ezrin/radixin/moesin-binding phosphoprotein 50 is regulated by PKA.</a> Proc. Natl. Acad. Sci. USA. 100, 342-346 (1073).</li><li>Li, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysophosphatidic+acid+inhibits+cholera+toxin-induced+secretory+diarrhea+through+CFTR-dependent+protein+interactions.">Lysophosphatidic acid inhibits cholera toxin-induced secretory diarrhea through CFTR-dependent protein interactions.</a> J. Exp. Med. 202, 975-986 (2005).</li><li>Li, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+coupling+of+cAMP+transporter+to+CFTR+chloride+channel+function+in+the+gut+epithelia.">Spatiotemporal coupling of cAMP transporter to CFTR chloride channel function in the gut epithelia.</a> Cell. 131, 940-951 (2007).</li><li>Li, C., Schuetz, J. D., Naren, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+carcinogen+NNK+transporter+MRP2+regulates+CFTR+function+in+lung+epithelia:+implications+for+lung+cancer.">Tobacco carcinogen NNK transporter MRP2 regulates CFTR function in lung epithelia: implications for lung cancer.</a> Cancer Lett. 292, 246-253 (2010).</li><li>Hall, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+C-terminal+motif+found+in+the+beta2-adrenergic+receptor,+P2Y1+receptor+and+cystic+fibrosis+transmembrane+conductance+regulator+determines+binding+to+the+Na+/H++exchanger+regulatory+factor+family+of+PDZ+proteins.">A C-terminal motif found in the beta2-adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the Na+/H+ exchanger regulatory factor family of PDZ proteins.</a> Proc. Natl. Acad. Sci. U.S.A. 95, 8496-8501 (1998).</li><li>Short, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+apical+PDZ+protein+anchors+the+cystic+fibrosis+transmembrane+conductance+regulator+to+the+cytoskeleton.">An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton.</a> J. Biol. Chem. 273, 19797-19801 (1998).</li><li>Wang, S., Yue, H., Derin, R. B., Guggino, W. B., Li, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accessory+protein+facilitated+CFTR-CFTR+interaction,+a+molecular+mechanism+to+potentiate+the+chloride+channel+activity.">Accessory protein facilitated CFTR-CFTR interaction, a molecular mechanism to potentiate the chloride channel activity.</a> Cell. 103, 169-179 (2000).</li><li>Sun, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E3KARP+mediates+the+association+of+ezrin+and+protein+kinase+A+with+the+cystic+fibrosis+transmembrane+conductance+regulator+in+airway+cells.">E3KARP mediates the association of ezrin and protein kinase A with the cystic fibrosis transmembrane conductance regulator in airway cells.</a> J. Biol. Chem. 275, 29539-29546 (2000).</li><li>Cheng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Golgi-associated+PDZ+domain+protein+modulates+cystic+fibrosis+transmembrane+regulator+plasma+membrane+expression.">A Golgi-associated PDZ domain protein modulates cystic fibrosis transmembrane regulator plasma membrane expression.</a> J. Biol. Chem. 277, 3520-3529 (1074).</li><li>Scott, R. O., Thelin, W. R., Milgram, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+PDZ+protein+regulates+the+activity+of+guanylyl+cyclase+C,+the+heat-stable+enterotoxin+receptor.">A novel PDZ protein regulates the activity of guanylyl cyclase C, the heat-stable enterotoxin receptor.</a> The Journal of biological chemistry. 277, 22934-22941 (1074).</li><li>Lee, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+regulation+of+cystic+fibrosis+transmembrane+conductance+regulator+by+competitive+interactions+of+molecular+adaptors.">Dynamic regulation of cystic fibrosis transmembrane conductance regulator by competitive interactions of molecular adaptors.</a> The Journal of biological chemistry. 282, 10414-10422 (2007).</li><li>Gee, H. Y., Noh, S. H., Tang, B. L., Kim, K. H., Lee, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+DeltaF508-CFTR+trafficking+via+a+GRASP-dependent+unconventional+secretion+pathway.">Rescue of DeltaF508-CFTR trafficking via a GRASP-dependent unconventional secretion pathway.</a> Cell. 146, 746-760 (2011).</li><li>Naren, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+study+of+intermolecular+and+intramolecular+interactions+regulating+CFTR+function.">Methods for the study of intermolecular and intramolecular interactions regulating CFTR function.</a> Met. Molecul. Med. 70, 175-186 (2002).</li><li>Li, C., Roy, K., Dandridge, K., Naren, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+assembly+of+cystic+fibrosis+transmembrane+conductance+regulator+in+plasma+membrane.">Molecular assembly of cystic fibrosis transmembrane conductance regulator in plasma membrane.</a> The Journal of biological chemistry. 279, 24673-24684 (2004).</li><li>Li, C., Naren, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+CFTR+Interactome+in+the+Macromolecular+Complexes.">Analysis of CFTR Interactome in the Macromolecular Complexes.</a> Met. Molecul. Med. 741, 255-270 (2011).</li><li>Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemokine+receptor+CXCR2+macromolecular+complex+regulates+neutrophil+functions+in+inflammatory+diseases.">A chemokine receptor CXCR2 macromolecular complex regulates neutrophil functions in inflammatory diseases.</a> J. Biol. Chem. , (2011).</li></ol>

Bu protokolde Bu şekilde, daha önce 16-19,29,30 bildirilen in vitro monte edilmesi ve saflaştırılmış protein (ya da proteininin parçacıkları) ve / veya hücre lizatları kullanılarak makromoleküler sinyalleşme kompleksi ihtiva eden bir CFTR tespiti için bir yöntemi göstermektedir. Hazırlık sürecinde iyi sonuçlar aşağıdaki kritik noktalar elde etmek için özel dikkat gerektirir: <ul><li> Bu işlem tampon maddesi ile pH adım 1) &#39;de anlatıldığı gibi GST-füzyon prote...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> pGEX4T-1 vektör </td><td> GE Healthcare </td><td> 28-9545-49 </td><td> Eskiden Amersham Biosciences </td></tr><tr><td> pMAL-C2 vektör </td><td> New England Biolabs </td><td></td><td></td></tr><tr><td> pET30 vektör </td><td> Merck Kimyasallar </td><td> 69077-3 </td><td> Eskiden Novagen </td></tr><tr><td> Glutatyon agaroz boncuk </td><td> BD Biosciences </td><td> 554780 </td><td></td></tr><tr><td> Amiloz reçine </td><td> New England Biolabs </td><td> E8021S </td><td></td></tr><tr><td> Talon boncuk </td><td> Clontech&#39;in </td><td> 635501 </td><td></td></tr><tr><td> indirgenmiş glutatyon </td><td> BD Biosciences </td><td> 554782 </td><td></td&gt; </tr><tr><td> imidazol </td><td> Balıkçı </td><td> BP305-50 </td><td></td></tr><tr><td> maltoz </td><td> Balıkçı </td><td> BP684-500 </td><td></td></tr><tr><td> G-proteinine agaroz </td><td> Merck Kimyasallar </td><td> 69704-3 </td><td> Eskiden Novagen </td></tr><tr><td> Anti-Flag HRP </td><td> Sigma </td><td> A8592 </td><td></td></tr><tr><td> Anti-CFTR IgG </td><td> Ismarlama </td><td> R1104 </td><td> aa 722-734 de CFTR epitop tanıma mAb </td></tr><tr><td> Anti-MRP2 IgG </td><td> Chemicon Uluslararası </td><td> MAB4148 </td><td> Millipore şimdi bir parçası </td></tr></tbody></table> Tablo 2. Özel reaktifleri ve ekipmanları.

in vitro analysis of pdz-dependent cftr macromolecular signaling complexes

Kistik fibrozis transmembran kondüktans regülatörü (CFTR), öncelikle epitel hücrelerinin apikal hücre zarlarında bulunan bir klor kanalı, 1-3 transepitelyal sıvı dengesinin önemli bir rol oynar. Kistik fibrozis (CF) 4 ve sekretuar ishal 5: CFTR iki önemli hastalıklarla ilişkisi olmuştur. CF, CFTR Cl-kanalının sentez ya da işlevsel aktivitesini azalır. Bu hastalık Amerika Birleşik Devletleri&#39;nde 2500 Kafkasyalılar 6 yaklaşık 1 etkiler. Aşırı CFTR etkinliği de cAMP veya bağırsak 7 cGMP üretimini uyarır toksin kaynaklı sekretuar ishal (örneğin, kolera toksini ve ısıya dayanıklı enterotoksin tarafından E. coli) durumlarında ortaya konmuştur. Biriken deliller CFTR ve taşıyıcılar, iyon kanalları, reseptörler, kinazlar, fosfatazlar, sinyal dahil olmak üzere diğer proteinler giderek artan sayıda, arasındaki fiziksel ve fonksiyonel etkileşimlerin varlığını aklaING molekülleri ve sitoskeletal elemanları ve CFTR ve bağlayıcı protein arasındaki bu etkileşimlerin kritik in vitro ve in vivo da 8-19 CFTR-aracılı transepitelyal iyon taşıma düzenleyen dahil edilmesi gösterilmiştir. Bu protokol, biz sadece yöntemleri üzerinde durulacak bir protein bağlayan bir motif sahip CFTR karboksil terminal kuyruk arasındaki etkileşimlerin çalışmada yardım ve [motifi PSD95/Dlg1/ZO-1 (PDZ) olarak anılacaktır] Belirli bir bağlayıcı modülü ihtiva iskelesi proteinlerin grubu PDZ etki olarak anılacaktır. Şimdiye kadar, birkaç farklı PDZ iskele proteinler gibi NHERF1 gibi çeşitli akrabalıklar, NHERF2, PDZK1, PDZK2, CAL (CFTR ilişkili ligand), Shank2 ile CFTR karboksil terminal kuyruk bağlamak ve 20-27 ÇIKAN bildirilmiştir. PDZ iskele proteinler tarafından tanınır CFTR içinde PDZ motifi C terminus (yani, insan KFTR 1477-DTRL-1480) 20 son dört amino asitler olduğu. İlginç bir şekilde,CFTR akrabalıklar 22 farklı da olsa, NHERFs ve PDZK1 hem birden fazla PDZ alanı bağlayabilirsiniz. CFTR bağlayıcı bakımından Bu çok-değerliğine PDZ iskelesi proteinleri hücreleri 16-18 seçici / spesifik ve verimli bir sinyalleşme için kompleksleri sinyalleşme CFTR makromoleküler oluşumunu kolaylaştıran olabileceğini düşündürmektedir işlevsel bir öneme sahip olduğu gösterilmiştir. Çoklu biyokimyasal testler incelemek için geliştirilmiştir CFTR-içeren bu işbirliğinin immünopresipitasyon, açılan tayini, çifti-bilge birleştirme testi, kolorimetrik çifti-bilge birleştirme testi ve makromoleküler karmaşık bir montaj test 16-19,28,29 gibi protein etkileşimleri, . Burada protein-protein ya da CFTR 16-19,28,29 içeren etki alanı etki alanı etkileşimlerini incelemek için laboratuvar tarafından yaygın olarak kullanılmaktadır in vitro, karmaşık bir PDZ motifi bağımlı CFTR içeren makromoleküler montaj ayrıntılı yordamlar odaklanın.

Watch this Scientific Journal Video about In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes at JoVE.com

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Kistik fibrozis transmembran kondüktans regülatörü (CFTR), epitelyal bir klor kanalı, çeşitli proteinler ile etkileşim ve önemli hücre süreçlerini düzenleyen bildirilmiştir; aralarında CFTR PDZ motifi aracılı etkileşimleri de belgelenmiştir. Bu protokol bizim karmaşık bir sinyal PDZ bağımlı CFTR makromoleküler birleştirmek için geliştirilen yöntemler açıklanır<em&gt; In vitro</em&gt;.

PDZ bağımlı CFTR makromoleküler Sinyal Kompleksleri İn Vitro Analizi

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

Research

JoVE Journal

Biology

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann-astrosit Etkileşimleri Analizi In Vitro Tahliller

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

Efektör-Kompleksleri kullanarak BMP4 Mansabında Sinyal Algılama In situ PLA, Proximity ligasyon Testi

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Saç Rejenerasyon sırasında epitelyal-mezenkimal Sinyal Rapid Genetik Analizi

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

İfade Klonlama tarafından Grb2 Sinyal Kompleksi Eğitim için bir Yüksek içerik Görüntüleme İş Akışı

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Hücre Tek katmanlar mekanik uyarılması bağlı Kalsiyum Dalga Yayılım: Sığır Kornea Endotel Hücreleri Örneği

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Mukayeseli Proteomik Analizi Metabolik Etiketleme ve Membran Fraksiyonasyon<em&gt; Arabidopsis thaliana</em&gt; Süspansiyon Hücre Kültürleri

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

İzolasyon ve Kültür Yetişkin Fare Hücre Sinyal için kardiyomiyositlerin ve<em&gt; In vitro</em&gt; Kalp hipertrofisi

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolik Oranı Ölçümü<em&gt; Drosophila</em&gt; Respirometrik kullanarak

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

Bitkiler ve Yeşil Alg Işık hasat Kompleksleri İn Vitro Sulandırma içinde

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

Saf Yabanitip ve Mutant CFTR Protein Fonksiyonel Sulandırma ve Kanal Aktivite Ölçümleri

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...