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 Stunden. </li></ol></li><li> Herstellung von Zellmembranfraktionen und Kernextrakten <ol><li> Wasche die Zellen einmal mit PBS, schaben in 1 ml PBS und Zentrifugation bei 1.000 xg für 5 min bei 4 ° C. </li><li> Die Zellen in einem eiskalten Puffer , enthaltend 10 mM HEPES-KOH (pH 7,6), 10 mM KCl, 1,5 mM MgCl 2, 1 mM Natrium - Ethylendiamintetraessigsäure (EDTA), 1 mM Natrium Ethylenglycol tetraessigsäure (EGTA), 250 mM Saccharose, und eine Mischung von Proteaseinhibitoren (5 μg / ml Pepstatin A, 10 &amp; mgr; g / ml Leupeptin 0,5 mM Phenylmethylsulfonylfluorid (PMSF), 1 mM Dithiothreitol (DTT) und 25 ug / ml N-Acetyl-Leu-Leu-Norleu-al (ALLN)) für 30 min auf Eis. </li><li> Passieren Zellextrakten durch eine 22 G x 1 1/2 Nadel 30mal und Zentrifuge bei 890 × g bei 4 ° C für 5 min, zu isolieren Kernen. </li><li> Verwenden Sie den Überstand zur Abtrennung von Membranfraktionen (Schritt 1.2.7). Resuspendieren Kernpellet in 0,1 ml Puffer C (20 mM HEPES / KOH pH 7,6, 0,42 M NaCl, 2,5% (v / v) Glycerol, 1,5 mM MgCl 2, 1 mM Natrium - EDTA, 1 mM Natrium - EGTA und eine Mischung aus Protease-Inhibitoren (5 ug / ml Pepstatin A, 10 &amp; mgr; g / ml Leupeptin, 0,5 mM PMSF, 1 mM DTT, und 25 &amp; mgr; g / ml ALLN)). </li><li> Drehen, um die Suspension bei 4 ° C für 60 min und zentrifugieren bei 20.000 xg in einer Mikrozentrifuge für 20 min bei 4 ° C. Der Überstand wird als &quot;Kernextrakte&quot; bezeichnet. </li><li> Erhitzen Sie die Kernextrakten bei 100 ° C für 10 min mit 5-fach-Ladepuffer (312,5 mM Tris-HCl pH 6,8, 10% SDS, 50% Glycerin, 12,5% β-Mercaptoethanol und 0,05% Bromphenolblau) vor einer SDS-PAGE unterzogen wird. </li><li> Zentrifugieren Sie den Überstand von der ursprünglichen 890 × g Spin bei 20.000 × g für 20 min bei 4 ° C. Für die anschließende Western Blot-Analyse, um das Pellet in 0,1 ml SDS-Lyse-Puffer (10 mM Tris-HCl pH 6,8, 100 mM NaCl, 1% (v / v) Natriumdodecylsulfat (SDS), 1 mM Natrium-EDTA, lösen und 1 mM Natrium EGTA). Dies wird als &quot;Membranfraktion&quot; bezeichnet. HINWEIS: Wir verwenden 20.000 × g für 20 min Membranen zu pelletieren. 100.000 × g für 10 - 20 min wurde in den Zeitungen von der Brown und Goldstein Labor 15 verwendet. </li><li> Inkubieren der Membranfraktion bei 37 ° C für 30 min und bestimmen, um die Proteinkonzentration durch Bradford-Protein-Assay. 1 &amp; mgr; l 100x Bromphenolblau-Lösung, bevor die Proben SDS-PAGE unterzogen wird. </li><li> Last 25-50 ug Gesamt - Membranproteine auf einem 10% SDS-PAGE - Gel 39. Führen Sie das Gelbei 80 V für 15 min, dann auf 130 V zu ändern und für eine weitere 115 min laufen. Übertragen bei 140 mA für 130 min 39. <ol><li> Für die Western-Blot-Analyse, verwenden anti-SCAP Antikörper die gesamte SCAP Protein nachzuweisen. Verwenden Protein (PDI), ein ER- lokalisiertes Protein 40, als eine interne Kontrolle. Verwenden anti-SREBP-1-Antikörper die N-terminale Bande von SREBP-1 zu erkennen. Verwenden Sie Lamin A als interne Kontrolle für Kernextrakten 31. </li></ol></li></ol></li></ol> 2. Analyse der SCAP N -glycosylation in menschlichen Zellen <ol><li> Zellkultur, Transfektion und Behandlung <ol><li> Für Membrananalyse, seed ~ 1 × 10 6 HEK293T - Zellen in einer 10 cm - Schale mit DMEM ergänzt mit 5% FBS und Inkubieren der Zellen bei 37 ° C und 5% CO 2 für 24 Stunden vor der Transfektion. </li><li> Verdünnen Sie 4 - 8 ug GFP-SCAP - Plasmid (ein Geschenk von Dr. Peter Espenshade) 22 in 1 ml Serum Medium reduziert und vorsichtig mischen. </li><li> In 8-16 &amp; mgr; l DNA-Transfektionsreagenz durch direktes Pipettieren in die 1 ml reduziert Serum Medium Plasmide enthalten, und vorsichtig mischen. </li><li> Inkubieren des Transfektionsreagenz / Plasmid-Komplex für 25 min bei Raumtemperatur. </li><li> Hinzufügen Transfektionskomplex zu den Zellen mit frischem Medium und weiterhin Kultur für 24 Stunden bei 5% CO 2 und 37 ° C. </li><li> Wasche einmal mit PBS und zu behandeln , die Zellen für 12 h mit oder ohne Glucose (5 mM) in Gegenwart oder Abwesenheit von Tunicamycin (1 ug / ml), eine n -glycosylation Inhibitor. </li></ol></li><li> Bereiten Sie Zellmembran Pellets wie in den Schritten 1.2.1 beschrieben, 1.2.2, 1.2.3 und 1.2.7. </li><li> Der Nachweis von SCAP N -glycosylation <ol><li> Trypsin Proteolyse <ol><li> Resuspendieren der Membranpellets aus Zellen GFP-SCAP in 114 ul Puffer , enthaltend 10 mM HEPES · KOH (pH 7,4) überexprimieren, 10 mM KCl, 1,5 mM MgCl 2, 1 mM Natrium - EDTA und 100 mM NaCl. </li><li> Inkubiere 5781; l Aliquots von Membranproteinen in Abwesenheit oder Anwesenheit von 1 &amp; mgr; l Trypsin (1 ug / ul) in einem Gesamtvolumen von 58 &amp; mgr; l für 30 min bei 30 ° C. </li><li> Stop Reaktionen durch Zugabe von 2 ul (400 Einheiten) von Sojabohnen-Trypsin-Inhibitor. </li></ol></li><li> Deglycosylation von Peptid- N- Glycosidase F (PNGase F) <ol><li> Für eine nachfolgende Behandlung mit PNGase F, mit 10 ul Lösung mit 3,5% (wt / vol) SDS und 7% (Vol / Vol) 2-Mercaptoethanol zu jeder Probe enthält. </li><li> Nach dem Erhitzen bei 100 ° C für 10 min, fügen sequentiell 7 ul 0,5 M Natriumphosphat (pH 7,5), 7 ul 10% (v / v) Nonidet P-40 mit 12 × Protease-Inhibitoren, und 2 &amp; mgr; l (0,5 U / &amp; mgr; l) von PNGase F. </li><li> Nach Inkubation bei 37 ° C für 3 h, Stopp Reaktionen durch die Zugabe von 5x Ladepuffer (312,5 mM Tris-HCl pH 6,8, 10% SDS, 50% Glycerin, 12,5% β-Mercaptoethanol und 0,05% Bromphenolblau). Erhitzen Sie die Mischungen bei 100 ° C für 10 min und nach SDS-PAGE. Last 25-50 ug Gesamt - Membranproteine auf einem 10% SDS-PAGE - Gel 39. Führen Sie das Gel bei 80 V für 15 min, dann auf 130 V für einen weiteren 115 min ändern. Übertragen bei 140 mA für 130 min. </li><li> Für die Western - Blot, verwenden 10 ug / ml anti-SCAP (9D5) Antikörper -glycosylation N und Gesamt SCAP Protein nachzuweisen. </li></ol></li></ol></li></ol> 3. Nachweis von SCAP Handel von ER zum Golgi durch Verwendung von GFP-SCAP in menschlichen Zellen <ol><li> Seed 2,5 × 10 5 U87 - Zellen in einer 60 mm Schale mit DMEM ergänzt mit 5% FBS und Inkubieren der Zellen bei 37 ° C und 5% CO 2 für 24 h vor der Transfektion. </li><li> Verdünnen Sie 4 ug GFP-SCAP-Plasmid in 0,5 ml reduziert Serum Medium und mischen. </li><li> In 8 ul DNA-Transfektionsreagenz durch Pipettieren direkt in 0,5 ml reduziert Serum Medium Plasmide enthalten, und vorsichtig mischen. </li><li> Inkubieren des Transfektionsreagenz / Plasmid-Komplex für 25 min bei RaumTemperatur. </li><li> Hinzufügen Transfektionskomplex zu den Zellen mit frischem Medium und weiterhin Kultur für 24 Stunden bei 5% CO 2 und 37 ° C. </li><li> Reseed 5 - 10 × 10 4 Zellen in einer 6-Well - Platte , die eine Glasdeckglas (22 mm × 22 mm) und weiter zur Kultur bei 37 ° C und 5% CO 2 für 24 Stunden enthält. </li><li> Wasche einmal mit PBS und zu behandeln, die Zellen für 12 h mit oder ohne Glucose (5 mM) in Gegenwart oder Abwesenheit von Tunicamycin (1 ug / ml). </li><li> Waschen Sie die Zellen einmal mit PBS und fixieren in 4% Formaldehyd für 10 min. </li><li> Waschen Sie die Zellen dreimal mit PBS, kehren Sie die Deckgläser, montieren Sie auf Objektträger zusammen mit Antifade Reagenz und versiegeln Sie die Deckgläser mit Nagellack. </li><li> Überprüfen Sie die GFP-SCAP Handel mit einem 63X -Ölimmersionsobjektiv in einem Laser - Scanning - konfokalen Mikroskop 41. </li></ol>

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sugar+Makes+Fat+by+Talking+to+SCAP.">Sugar Makes Fat by Talking to SCAP.</a> Cancer Cell. 28, 548-549 (2015).</li><li>Guo, 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=EGFR+signaling+through+an+Akt-SREBP-1-dependent,+rapamycin-resistant+pathway+sensitizes+glioblastomas+to+antilipogenic+therapy.">EGFR signaling through an Akt-SREBP-1-dependent, rapamycin-resistant pathway sensitizes glioblastomas to antilipogenic therapy.</a> Sci Signal. 2, 82 (2009).</li><li>Guo, 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+LXR+agonist+promotes+GBM+cell+death+through+inhibition+of+an+EGFR/AKT/SREBP-1/LDLR-dependent+pathway.">An LXR agonist promotes GBM cell death through inhibition of an EGFR/AKT/SREBP-1/LDLR-dependent pathway.</a> Cancer Discov. 1, 442-456 (2011).</li><li>Guo, D., Bell, E. H., Mischel, P., Chakravarti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+SREBP-1-driven+lipid+metabolism+to+treat+cancer.">Targeting SREBP-1-driven lipid metabolism to treat cancer.</a> Curr Pharm Des. 20, 2619-2626 (2014).</li><li>Bell, E. H., Guo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarkers+for+malignant+gliomas.">Biomarkers for malignant gliomas.</a> Malignant Gliomas, Radiation Medicine Rounds. 3, 339-357 (2012).</li><li>Geng, F., 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=Inhibition+of+SOAT1+suppresses+glioblastoma+growth+via+blocking+SREBP-1-mediated+lipogenesis.">Inhibition of SOAT1 suppresses glioblastoma growth via blocking SREBP-1-mediated lipogenesis.</a> Clin Cancer Res. , (2016).</li><li>Ru, P., 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=Feedback+Loop+Regulation+of+SCAP/SREBP-1+by+miR-29+Modulates+EGFR+Signaling-Driven+Glioblastoma+Growth.">Feedback Loop Regulation of SCAP/SREBP-1 by miR-29 Modulates EGFR Signaling-Driven Glioblastoma Growth.</a> Cell Rep. , (2016).</li><li>Green, M. R., Sambrook, 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> Molecular Cloning: A Laboratory Manual (Fourth Edition). , (2012).</li><li>Uehara, 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=S-nitrosylated+protein-disulphide+isomerase+links+protein+misfolding+to+neurodegeneration.">S-nitrosylated protein-disulphide isomerase links protein misfolding to neurodegeneration.</a> Nature. 441, 513-517 (2006).</li><li>Miyashita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+microscopy+for+intracellular+co-localization+of+proteins.">Confocal microscopy for intracellular co-localization of proteins.</a> Methods Mol Biol. 1278, 515-526 (2015).</li><li>Cao, J., Guo, S., Arai, K., Lo, E. H., Ning, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+extracellular+signaling+utilizing+a+glycoproteomic+approach:+lectin+blot+surveys,+a+first+and+important+step.">Studying extracellular signaling utilizing a glycoproteomic approach: lectin blot surveys, a first and important step.</a> Methods Mol Biol. 1013, 227-233 (2013).</li></ol>

The authors have nothing to disclose.

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.

<table border="0" cellpadding="0" cellspacing="0" width="634">
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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<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 ...

analysis-of-scap-n-glycosylation-and-trafficking-in-human-cells

Research

JoVE Journal

Biology

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

8.6K Aufrufe.  The Ohio State University Comprehensive Cancer Center and College of Medicine.  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. 

Serie: Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Video Bioinformatics Analysis of Human Embryonic Stem Cell 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 software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

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

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

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 ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

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

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 include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

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

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

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

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and 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

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

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 is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

Here we present a protocol to image cells expressing green fluorescent protein-tagged angiotensin type 1a receptors during endocytosis initiated by angiotensin II treatment. This technique includes labeling lysosomes with a second fluorescent marker, and then utilizing software to analyze the co-localization of receptor and lysosome in three dimensions over time.

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

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of 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 ...

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions. However, due to the complexity of the process of SUMOylation in vivo, it is more challenging to detect and analyze protein SUMOylation in cells, especially when under endogenous conditions. A recent study by the authors of this paper revealed that endogenous retinoblastoma (Rb) protein, a tumor suppressor that is vital to the negative regulation of the cell cycle progression, is specifically SUMOylated at the early G1 phase. This paper describes a protocol for the detection and analysis of Rb SUMOylation under both endogenous and exogenous conditions in human cells. This protocol is appropriate for the phenotypical and functional investigation of the SUMO-modification of Rb, as well as many other SUMO-targeted proteins, in human cells.

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.

In Vivo Detektion und Analyse von Rb Protein Sumoylierung in menschlichen Zellen

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, ...

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