Name: analysis of scap n-glycosylation and trafficking in human cells
Uploaded: 2016-11-08T14:00:00
Description: Watch this Scientific Journal Video about Analysis of SCAP N-glycosylation and Trafficking in Human Cells at JoVE.com

We are grateful to Drs. Mike S. Brown and Joseph L. Goldstein for their agreement to publish this method according to the methods described in their publications. We appreciate Dr. Peter Espenshade for sharing the GFP-SCAP plasmid. This work was supported by NIH grants NS072838 and NS079701 to D.G., American Cancer Society Research Scholar Grant RSG-14-228-01-CSM to D.G., and OSUCCC Pelotonia Postdoctoral Fellowship to C.C. We also appreciate the support from the Ohio State Neuroscience Core (P30 NS038526) and OSUCCC Translational Therapeutic Program seed grant and start-up funds to D.G.

İnsan hücrelerinde 1. Tespit endojen SCAP Protein <ol><li> Hücre Kültürü ve İşlem <ol><li> Tohum yaklaşık 1 x 10 6 U87% 5 fetal sığır serumu (FBS) ile desteklenmiş Dulbecco Modifiye Edilmiş Eagle Ortamı (DMEM) ile 10 cm&#39;lik bir tabak hücreleri ve tedaviden önce 24 saat boyunca CO2, 37 ° C&#39;de inkübe hücreleri ve% 5. </li><li> fosfat tamponlu tuzlu su (PBS) ile bir kez hücreler yıkanır, daha sonra 12 saat boyunca (5 mM) ile birlikte ya da glükoz ihtiva etmeyen taze ...

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Bu çalışmada, insan hücrelerinde membran fraksiyonlarının izolasyonu için ve Western blot ile SCAP N -glycosylation ve toplam proteinin belirlenmesi için numune hazırlanması için bir protokol açıklar. Biz daha GFP-etiketleme ve konfokal mikroskopi kullanılarak SCAP kaçakçılığı izlemek için bir yöntem sağlar. Yöntem özellikle membran proteini analiz etmek için kullanılan ve SCAP N -glycosylation ve insan ticaretini soruşturma önemli bir araçtır. <p class="jove_content"...

Lipid metabolizması deregülasyon kanseri ve metabolizma hastalıkları 1-6 ortak bir özelliğidir. Bu işlemlerde, sterol düzenleyici elementi bağlayan proteinler (SREBPs), transkripsiyon faktörleri ailesidir, alım ve kolesterol, yağlı asitlerin sentezi ve fosfolipid 7-10 önemli genlerin ekspresyonunu kontrol etmede çok önemli bir rol oynar. SREBP-1 a, SREBP-1 c ve SREBP-2 de dahil olmak üzere SREBPs, iki transmembran 7 sayesinde endoplazmik retikulum (ER) zarına bağlanan etkin olmayan öncüler olarak sentezlenir. SREBPs N-terminali, hedef genlerinin 11 transaktivasyonu için bir DNA-bağlama alanını içerir. SREBP öncülerinin C-terminali SREBP bölünmesi aktive edici protein (SCAP) 12,13, SREBP stabilitesi ve aktivasyon 14-16 düzenlenmesinde çok önemli bir rol oynayan bir politopik membran proteinine bağlanır. implementranskripsiyonel fonksiyonunun yon iki proteazlar ardışık SREBP ayrılması ve daha sonra lipojenik genlerin 7 transaktivasyonu için çekirdeğin içine girer N-terminal fragmanı serbest Golgi, ER SCAP / SREBP kompleksinin, gerektirir. Bu işlemlerde, ER membran sterol seviyeleri ER 7,17 den SCAP / SREBP kompleksinin çıkış kontrol eder. Yüksek sterol koşullar altında, sterol Scap veya ER yerleşik bağlanan insülin ile uyarılan gen proteini-1 (INSIG-1) veya 2 (INSIG-2), içinde SCAP / SREBP kompleksi korumak Insigs ile Scap ilişkisini arttırıcı ER 18-20. sterol düzeyleri azalma olduğunda, SCAP Insigs ile ayrışmaktadır. Bu ortak kaplama proteinleri (COP) II kompleksi ile SCAP etkileşim sağlayan bir SCAP konformasyonel değişime yol açar. Karmaşık tomurcuklanma vezikülleri içine SCAP / SREBP kompleksi dahil edilmesine aracılık eder ve Golgi 21,22 ER; nakliye yönlendirir. TRANSLOC üzerineGolgi ation, SREBPs ardışık N-terminali 7,23-29 serbest kalmasına yol, alana, 1 ve-2 proteaz tarafından ayrılmaktadır. SCAP proteini N asparajinde oligosakkaritler -bağlı üç taşır (N) N263, N590, N641 ve 15 konumlandırır. Biz son zamanlarda bu sitelerde SCAP glukoz aracılı N -glycosylation düşük sterol koşullarında 30-32 altında Golgi ER SCAP / SREBP kaçakçılığı için bir ön koşul olduğunu ortaya koymuştur. (NNN qqq için) glutamin için üç asparagin mutasyon yoluyla SCAP glikosilasyon kaybı SCAP protein ve SREBP aktivasyon 31 azaltılması istikrarsızlık SCAP / SREBP kompleksinin ve sonuçların ticaretini devre dışı bırakır. Bizim son veriler de bu SREBP-1 yüksek glioblastoma aktive ve SCAP N -glycosylation 4,31,33,34 tarafından düzenlenir göstermektedir. Hedefleme SCAP / SREBP-1 sinyal maligniteler ve metabolik s tedavisinde yeni bir strateji olarak ortaya çıkmaktadır1,3,35-38 yndromes. Bu nedenle, SCAP proteini ve N -glycosylation düzeylerini analiz etmek ve insan hücreleri ve hasta dokularda kaçakçılığı izlemek için etkin bir yöntem geliştirmektir önemlidir. ER &#39;in içinde SCAP proteini (amino asitler 540-707) luminal bölgesi sağlam zarları tripsin 15 ile muamele edildiğinde proteoliz korunur iki N -glycosylation sitesi (N590 ve N641) içerir. Bu lümen parçası, sodyum dodesil sülfat poliakrilamid jel elektroforezi (SDS-PAGE) 15,31 ile Scap bireysel glikosile varyantlarının çözünürlüğünü sağlamak için yeterince küçüktür ~ 30 kDa&#39;lık bir moleküler ağırlığa sahiptir. Burada, N Scap arasında -glycosylation ve insan toplam hücre proteini tespit etmek için bir yöntem sağlar. Bu protokol, Brown ve Goldstein, laboratuarda 15 ve son yayın 31 &#39;den yayınlarda açıklanan yönteme elde edilir. Protokol t kullanılabilirO memeli hücrelerinden SCAP protein çalışma.

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analysis of scap n-glycosylation and trafficking in human cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Şekil 1, Western blot Glükoz uyarısına karşı tepki olarak insan glioblastoma U87 hücrelerinde endojen SCAP protein ve SREBP-1 nükleer formunun belirlenmesini göstermektedir. zar proteini SCAP &quot;zar fraksiyonu&quot; tespit edilir. SREBP-1 çekirdeği bir şekilde (N-terminal) &quot;Nükleer özler&quot; tespit edilir. SCAP protein düzeyi belirgin glükoz uyarılması ile geliştirilmiş ve SREBP-1 nükleer formu (KAE bir ER membran proteinlerinin iç k...

Watch this Scientific Journal Video about Analysis of SCAP N-glycosylation and Trafficking in Human Cells at JoVE.com

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Bu Western blot ile SCAP N -glycosylation ve toplam proteinin belirlenmesi için insan hücrelerinden membran fraksiyonu izolasyonu ve numune hazırlama yönelik değişik bir metot tarif etmektedir. Biz daha konfokal mikroskopi kullanılarak SCAP ticaretini izlemek için bir GFP-etiketleme yöntemi tanıtmak. Bu protokol, normal bir biyolojik laboratuvarlarda kullanılabilir.

Scap analizi<em&gt; K</em&gt; İnsan Hücreleri -glycosylation ve Ticareti

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of ...

Research

JoVE Journal

Biology

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Video Biyoinformatik İnsan Embriyonik Kök Hücre Colony Büyüme Analizi

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

Mürin Hematopoetik Kök Hücreler ve Lineage-kararlı progenitörlerin Fenotipik Analizi ve İzolasyon

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

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The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Mezenkimal Geçiş indüksiyon ve Epitel Analizi

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

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A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

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Kantitatif RT-PCR ile Bireysel İnsan Embriyonik Kök Hücreleri profilleme

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

Colocalizational Analizi Reseptör Sonrası içselleştirme Ticareti Uyuşturucu kaynaklı Değişiklikleri İzleme

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

Yüksek çözünürlüklü Time-lapse Görüntüleme ve Yaşayan İnsan Umbilikal Ven Endotelyal Hücrelerinde Mikrotubul Dinamiği Otomatik Analizi

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

Konfokal Mikroskobu kullanılarak Transfekte İnsan Embriyonik Böbrek Hücreleri Canlı Hücre Görüntüleme ve 3D Analiz Angiotensin Reseptör Tip 1a Ticareti

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Dinamik Hücresel Protrüzyonlarda Protein Konsantrasyonunun Yazılım Destekli Takibi için Grafiksel Bir Kullanıcı Arayüzü

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...