Name: analysis of scap n-glycosylation and trafficking in human cells
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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.

1. Nachweis von Endogene SCAP Protein in menschlichen Zellen <ol><li> Zellkultur und Behandlung <ol><li> Seed ~ 1 × 10 6 U87 - Zellen in einer 10 cm - Schale mit Dulbeccos modifiziertem Eagle Medium (DMEM) , supplementiert mit 5% fötalem Rinderserum (FBS) und Inkubation der Zellen bei 37 ° C und 5% CO 2 für 24 h vor der Behandlung. </li><li> Wasche die Zellen einmal mit phosphatgepufferter Kochsalzlösung (PBS), dann schaltet Zellen frisches DMEM-Medium mit oder ohne Glucose (5 mM) für 12 St...

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In dieser Studie beschreiben wir ein Protokoll für die Isolierung von Membranfraktionen in menschlichen Zellen und für die Herstellung der Proben für den Nachweis von SCAP N -glycosylation und Gesamtprotein durch Western - Blot verwendet wird . Wir bieten weiterhin ein Verfahren SCAP Handel zu überwachen, indem GFP-Markierung und der konfokalen Mikroskopie. Das Verfahren ist speziell verwendete Membranprotein zu analysieren und ist ein wichtiges Instrument SCAP N -glycosylation und Menschenhandel z...

Deregulation des Fettstoffwechsels ist ein gemeinsames Merkmal von Krebs und Stoffwechselerkrankungen 1-6. Bei diesen Verfahren Sterol regulatorische Element-bindende Proteine (SREBPs), eine Familie von Transkriptionsfaktoren, eine wichtige Rolle spielen , um die Expression von Genen , wichtig für die Aufnahme und die Synthese von Cholesterin, Fettsäuren bei der Kontrolle und Phospholipide 7-10. SREBPs, einschließlich SREBP-1a, SREBP-1c und SREBP-2, werden als inaktive Vorstufen synthetisiert, 7 durch zwei Transmembrandomänen an das endoplasmatische Retikulum (ER) -Membran binden. Der N-Terminus von SREBPs enthält eine DNA-Bindungsdomäne für die Transaktivierung von Zielgenen 11. Der C-Terminus von SREBP Vorläufer bindet an SREBP Spaltungs-aktivierendes Protein (SCAP) 12,13, einem polytopen Membranprotein , das eine zentrale Rolle in der Regulation der SREBP Stabilität und Aktivierung spielt 14-16. Die Umsettation der Transkriptionsfunktion erfordert die Translokation des SCAP / SREBP - Komplexes aus dem ER zum Golgi, wo zwei Proteasen sequentiell SREBP spalten und dessen N-terminales Fragment freizusetzen, die dann in den Kern 7 für die Transaktivierung von Genen lipogenic eintritt. Bei diesen Verfahren steuern die Pegel von Sterolen in der ER - Membran den Ausgang des SCAP / SREBP - Komplexes aus dem ER 7,17. Unter hohem Sterin Bedingungen bindet Sterin SCAP oder ER-resident Insulin-induzierte Gen-Protein-1 (Insig-1) oder -2 (Insig-2), die Verbesserung der Vereinigung von SCAP mit Insigs, die die SCAP / SREBP-Komplex in der Retain ER 18-20. Wenn Sterin-Spiegel senken, distanziert SCAP mit Insigs. Dies führt zu einer SCAP Konformationsänderung, die SCAP Wechselwirkung mit gemeinsamen Hüllproteine ​​(COP) II-Komplexes ermöglicht. Der Komplex vermittelt die Einbindung des SCAP / SREBP Komplexes in die angehenden Bläschen und lenkt seinen Transport aus dem ER zum Golgi 21,22. Nach TransLocation zum Golgi, SREBPs sequentiell von Standort 1 und Standort-2 - Proteasen gespalten, was zur Freisetzung des N-Terminus 7,23-29. SCAP Protein trägt drei N -verknüpftem Oligosaccharide an Asparagin (N) positioniert N263, N590 und N641 15. Wir vor kurzem ergeben , dass Glukose-vermittelten N -glycosylation von SCAP in diesen Seiten eine Vorbedingung ist für SCAP / SREBP Handel vom ER zum Golgi unter niedrigen Sterin Bedingungen 30-32. Der Verlust der SCAP Glykosylierung über Mutation aller drei Asparagin Glutamin (NNN zu QQQ) deaktiviert den Handel des SCAP / SREBP komplex und führt zur Instabilität der SCAP - Protein und die Verringerung der SREBP Aktivierung 31. Unsere jüngsten Daten zeigen auch , dass SREBP-1 ist stark in Glioblastoma aktiviert und reguliert durch SCAP N -glycosylation 4,31,33,34. Targeting SCAP / SREBP-1-Signalisierung zeichnet sich als eine neue Strategie malignen Erkrankungen und Stoffwechsel s zu behandelnyndromes 1,3,35-38. Daher ist es wichtig , eine effektive Methode zu entwickeln SCAP - Protein und N -glycosylation Ebenen und überwachen ihre Handels mit menschlichen Zellen und Geweben Patienten zu analysieren. Die luminalen Bereich von SCAP - Protein (Aminosäuren 540-707) in der ER enthält zwei N -glycosylation Stellen (N590 und N641) , die von Proteolyse geschützt sind , wenn intakten Membranen mit 15 Trypsin behandelt werden. Dieses luminalen Fragment ein Molekulargewicht von ~ 30 kDa , das klein genug ist , um die Auflösung der einzelnen glykosylierten Varianten SCAP durch Natriumdodecylsulfat - Polyacrylamid - Gelelektrophorese (SDS-PAGE) 15,31 zu ermöglichen. Hier stellen wir ein Verfahren N -glycosylation von SCAP und das Gesamtprotein in menschlichen Zellen zu detektieren. Dieses Protokoll wird von der Methode in den Publikationen von der Brown und Goldstein Labor beschrieben abgeleitet 15 und unsere jüngste Veröffentlichung 31. Das Protokoll kann in t verwendet werdener studieren von SCAP-Protein aus Säugerzellen.

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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">X-treme GENE HP DNA Transfection Reagent&#160;</td>
			<td style="width:163px;">Roche</td>
			<td style="width:123px;">6366236001</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEMI medium&#160;</td>
			<td>Life Technologies</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">trypsin&#160;</td>
			<td>Sigma</td>
			<td>T6567</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">soybean trypsin inhibitor</td>
			<td>Sigma</td>
			<td>T9777</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PNGase F&#160;</td>
			<td>Sigma</td>
			<td>P7367</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-SCAP (9D5) antibody&#160;&#160;</td>
			<td>Santa Cruz</td>
			<td>sc-13553</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GFP antibody&#160;</td>
			<td>Roche</td>
			<td>11814460001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SREBP-1 antibody (IgG-2A4)</td>
			<td>BD Pharmingen</td>
			<td>557036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PDI Antibody (H-17)</td>
			<td>Santa Cruz</td>
			<td style="width:123px;">sc-30932</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lamin A Antibody (H-102)</td>
			<td>Santa Cruz</td>
			<td style="width:123px;">sc-20680</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SCAP antibody (a.a 450-500)</td>
			<td>Bethyl Laboratories, Inc.</td>
			<td>A303-554A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) without glucose, pyruvate and glutamine&#160;&#160;</td>
			<td>Cellgro</td>
			<td>17-207-CV</td>
			<td>Add 1 Mm Pyravate and 4 mM Glutamine before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">22G x 1 1/2 needle&#160;</td>
			<td>BD&#160;</td>
			<td>305156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pepstatin A</td>
			<td>Sigma</td>
			<td>P5318</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">leupeptin</td>
			<td>Sigma</td>
			<td>L2884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PMSF</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DTT</td>
			<td>Sigma</td>
			<td>43819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ALLN</td>
			<td>Sigma</td>
			<td>A6185</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrocellulose membrane</td>
			<td>GE</td>
			<td>RPN3032D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA Solution 0.5 M PH8.5 100 ml&#160;</td>
			<td>VWR</td>
			<td>82023-102&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA 0.5 M sterile (PH 8.0) 50 ml</td>
			<td>Fisher Scentific</td>
			<td>50255956</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HyClone FBS</td>
			<td>Thermo scientific</td>
			<td>SH3007103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-mercaptoethanol&#160;</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bromophenol blue</td>
			<td>Sigma</td>
			<td>B8026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">prolong gold antifade reagent with dapi</td>
			<td>life technologies</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine (200 mM)</td>
			<td>life technologies</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium pyruvate</td>
			<td>life technologies</td>
			<td>11360-070</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Figur 1 zeigt den Nachweis von endogenem SCAP Protein und SREBP-1 Kernform in humanen Glioblastom U87 - Zellen als Reaktion auf Glucose - Stimulation durch Western - Blot verwendet wird . Das Membranprotein SCAP ist in der &quot;Membranfraktion&quot; detektiert. Der Kern Form (N-terminal) von SREBP-1 ist in den &quot;Kernextrakten&quot; erkannt. Die Höhe der SCAP Protein (PDI dient als eine interne Kontrolle der ER-Membran-Proteine) und SREBP-1 Kernform werden dur...

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

Wir beschreiben eine modifizierte Methode zur Membranfraktion isoliert von menschlichen Zellen und die Probenvorbereitung für den Nachweis von SCAP N -glycosylation und Gesamtprotein durch Western - Blot verwendet wird . Wir stellen ferner eine GFP-Markierungsverfahren SCAP Handel mittels konfokaler Mikroskopie zu überwachen. Dieses Protokoll kann in regelmäßigen biologischen Laboratorien verwendet werden.

Analyse von SCAP<em&gt; N</em&gt; -glycosylation Und Handel in menschlichen Zellen

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 bioinformatischen Analyse von humanen embryonalen Stammzellen Colony Growth

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 ...

Forschung

Biologie

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

Phänotypische Analyse und Isolierung von murinen hämatopoetischen Stammzellen und Lineage-festgelegte Vorläuferzellen

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

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

RNA-seq Analyse Transkriptomen Thrombin-behandelt und Control Human Pulmonary mikrovaskulären Endothelzellen

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

Induktion und Analyse von epithelialen zu mesenchymalen Transition

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

Effiziente Erzeugung Menschen induzierten pluripotenten Stammzellen aus menschlichen Körperzellen mit Sendai-Virus

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 ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Profiling Individuelle humanen embryonalen Stammzellen durch quantitative RT-PCR

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

Tracking-Drug-induzierte Veränderungen der Post-Rezeptor-Internalisierung Handel durch Colocalizational Analysis

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

Hochauflösende Zeitraffer-Imaging und automatische Analyse von Dynamik der Mikrotubuli in lebenden menschlichen Endothelzellen aus der Nabelvene

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

Live Cell Imaging und 3D-Analyse von Angiotensin-Rezeptor-Typ 1a Handel Transfizierte humane embryonale Nierenzellen mittels konfokaler Mikroskopie

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

Eine grafische Benutzeroberfläche für die softwaregestützte Verfolgung der Proteinkonzentration in dynamischen zellulären Vorsprüngen

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