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.

1. मानव कोशिकाओं में अंतर्जात की जांच SCAP प्रोटीन <ol><li> सेल संस्कृति और उपचार <ol><li> Dulbecco संशोधित ईगल मध्यम (DMEM) 5% भ्रूण गोजातीय सीरम (FBS) के साथ पूरक के साथ एक 10 सेमी डिश में बीज ~ 1 × 10 6 U87 कोशिकाओं और उपचार से पहले 24 ?...

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इस अध्ययन में, हम मानव कोशिकाओं में झिल्ली अंशों के अलगाव के लिए और पश्चिमी धब्बा का उपयोग करके SCAP एन -glycosylation और कुल प्रोटीन का पता लगाने के लिए नमूने की तैयारी के लिए एक प्रोटोकॉल का वर्णन है। हम आगे GFP ल?...

लिपिड चयापचय की ढील कैंसर और चयापचय रोगों 1-6 की एक आम लक्षण है। इन प्रक्रियाओं में, स्टेरोल नियामक तत्व बाध्यकारी प्रोटीन (SREBPs), प्रतिलेखन कारक के एक परिवार, तेज और कोलेस्ट्रॉल, फैटी एसिड के संश्लेषण, और फॉस्फोलिपिड 7-10 के लिए महत्वपूर्ण जीनों की अभिव्यक्ति को नियंत्रित करने में एक महत्वपूर्ण भूमिका निभाते हैं। SREBP -1 ए, SREBP -1 सी, और SREBP -2 सहित SREBPs, निष्क्रिय व्यापारियों है कि दो transmembrane डोमेन 7 के आधार पर जालिका (ईआर) झिल्ली के लिए बाध्य के रूप में संश्लेषित कर रहे हैं। SREBPs के एन टर्मिनस लक्ष्य जीन 11 की transactivation के लिए एक डीएनए बाध्यकारी डोमेन होते हैं। SREBP व्यापारियों की सी टर्मिनस SREBP दरार सक्रिय प्रोटीन (SCAP) 12,13, एक polytopic झिल्ली प्रोटीन है कि SREBP स्थिरता और सक्रियण 14-16 के नियमन में एक निर्णायक भूमिका निभाता को बांधता है। implemenट्रांसक्रिप्शनल समारोह के tation Golgi, जहां दो proteases क्रमिक रूप से SREBP फोड़ना और उसके एन टर्मिनल टुकड़ा है, जो तब lipogenic जीन 7 की transactivation के लिए नाभिक में प्रवेश करती जारी करने के लिए ईआर से SCAP / SREBP परिसर के translocation की आवश्यकता है। इन प्रक्रियाओं में, ईआर झिल्ली में sterols के स्तर को ईआर 7,17 से SCAP / SREBP परिसर के बाहर निकलने के नियंत्रित करते हैं। उच्च sterol शर्तों के तहत, sterol SCAP या ईआर निवासी को बांधता है इंसुलिन प्रेरित जीन प्रोटीन -1 (Insig -1) या -2 (Insig -2), Insigs कि में SCAP / SREBP जटिल बनाए रखने के साथ SCAP के सहयोग को बढ़ाने ईआर 18-20। sterol का स्तर कम होता है, SCAP Insigs साथ विघटित। यह एक SCAP गठनात्मक परिवर्तन है, जो आम कोट प्रोटीन (पुलिस) द्वितीय परिसर के साथ बातचीत की अनुमति देता SCAP की ओर जाता है। जटिल नवोदित vesicles में SCAP / SREBP परिसर का समावेश मध्यस्थता और Golgi 21,22 करने के लिए ईआर से अपने परिवहन निर्देशन। transloc परGolgi को समझना, SREBPs क्रमिक रूप से साइट -1 और साइट -2 proteases से चिपके रहते हैं, के एन टर्मिनस 7,23-29 रिहाई के लिए अग्रणी। SCAP प्रोटीन तीन एन asparagine पर से जुड़े oligosaccharides किया जाता है (एन) N263, N590, N641 और 15 पदों। हमने हाल ही में पता चला है कि इन साइटों में SCAP की ग्लूकोज की मध्यस्थता एन -glycosylation कम sterol शर्तों के तहत 30-32 Golgi को ईआर से SCAP / SREBP की तस्करी के लिए एक शर्त शर्त है। Glutamine (एनएनएन QQQ करने के लिए) के लिए सभी तीन asparagine के उत्परिवर्तन के माध्यम से SCAP ग्लाइकोसिलेशन की हानि SCAP / SREBP जटिल और SCAP प्रोटीन और SREBP सक्रियण 31 की कमी की अस्थिरता में परिणामों की तस्करी निष्क्रिय करता है। हमारे हाल के आंकड़े यह भी कि SREBP-1 है अत्यधिक glioblastoma में सक्रिय और SCAP एन -glycosylation 4,31,33,34 द्वारा नियंत्रित किया जाता है प्रदर्शित करता है। लक्ष्य निर्धारण SCAP / SREBP -1 संकेतन एक नई रणनीति कैंसर और चयापचय रों इलाज के रूप में उभर रहा हैyndromes 1,3,35-38। इसलिए, यह SCAP प्रोटीन और एन -glycosylation स्तर का विश्लेषण और मानव कोशिकाओं और मरीज के ऊतकों में इसकी तस्करी को नजर रखने के लिए एक प्रभावी तरीका विकसित करने के लिए महत्वपूर्ण है। ईआर में SCAP प्रोटीन (अमीनो एसिड 540-707) के ल्यूमिनल क्षेत्र दो एन -glycosylation साइटों (N590 और N641) कि प्रोटियोलिसिस से संरक्षित कर रहे हैं जब बरकरार झिल्ली trypsin 15 के साथ व्यवहार कर रहे हैं शामिल हैं। इस ल्यूमिनल टुकड़ा ~ 30 केडीए के एक आणविक वजन है कि काफी छोटा SCAP के व्यक्तिगत ग्लाइकोसिलेटेड वेरिएंट का संकल्प सोडियम सल्फेट dodecyl जेल वैद्युतकणसंचलन (एसडीएस पृष्ठ) 15,31 द्वारा अनुमति देने के लिए है। यहाँ, हम Scap के एन -glycosylation और मानव कोशिकाओं में कुल प्रोटीन का पता लगाने के लिए एक तरीका प्रदान करते हैं। इस प्रोटोकॉल विधि ब्राउन और गोल्डस्टीन प्रयोगशाला 15 और हमारे हाल ही में प्रकाशन के 31 से प्रकाशनों में वर्णित से ली गई है। प्रोटोकॉल टी में इस्तेमाल किया जा सकतावह स्तनधारी कोशिकाओं से SCAP प्रोटीन का अध्ययन करते हैं।

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			<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>
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		<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.

चित्रा 1 पश्चिमी धब्बा का उपयोग करके उत्तेजना ग्लूकोज के जवाब में अंतर्जात SCAP प्रोटीन और मानव glioblastoma U87 कोशिकाओं में SREBP -1 परमाणु फार्म का पता लगाने से पता चलता है। झिल्ली प्रोटीन SCAP &quot;झ?...

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

हम पश्चिमी धब्बा का उपयोग करके SCAP एन -glycosylation और कुल प्रोटीन का पता लगाने के लिए मानव कोशिकाओं से झिल्ली अंश अलगाव और नमूना तैयार करने के लिए एक संशोधित विधि का वर्णन है। हम आगे confocal माइक्रोस्कोपी का उपयोग SCAP की तस्करी की निगरानी के लिए एक GFP लेबलिंग विधि का परिचय। इस प्रोटोकॉल नियमित रूप से जीव विज्ञान प्रयोगशालाओं में इस्तेमाल किया जा सकता है।

SCAP का विश्लेषण<em&gt; एन</em&gt; -glycosylation और मानव कोशिकाओं में तस्करी

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

वीडियो मानव भ्रूण स्टेम सेल कॉलोनी विकास के विश्लेषण बायोइनफॉरमैटिक्स

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

प्ररूपी और murine hematopoietic स्टेम सेल और वंश प्रतिबद्ध progenitors के विश्लेषण से अलगाव

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

थ्रोम्बिन इलाज में आरएनए - seq Transcriptomes का विश्लेषण और नियंत्रण मानव फेफड़े Microvascular endothelial कोशिकाओं

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

Mesenchymal संक्रमण के लिए प्रेरण और उपकला का विश्लेषण

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

सेंडाइ वायरस के साथ मानव दैहिक कोशिकाओं से कुशल उत्पादन मानव प्रेरित pluripotent स्टेम सेल

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

मात्रात्मक RT-पीसीआर से व्यक्ति मानव भ्रूण स्टेम कोशिकाओं की रूपरेखा

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 विश्लेषण द्वारा रिसेप्टर पोस्ट-internalization के अवैध व्यापार में दवा प्रेरित परिवर्तन पर नज़र रखने

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

उच्च संकल्प समय चूक इमेजिंग और जीवित मानव नाल की शिरा endothelial कोशिकाओं में microtubule गतिशीलता की स्वचालित विश्लेषण

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

लाइव सेल इमेजिंग और ट्रांसफ़ेक्ट मानव भ्रूण गुर्दे की कोशिकाओं में Angiotensin के 3 डी विश्लेषण रिसेप्टर टाइप 1 ए तस्करी confocal माइक्रोस्कोपी का उपयोग

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

डायनामिक सेलुलर प्रोट्र्रेशन में प्रोटीन एकाग्रता के सॉफ़्टवेयर-सहायता प्राप्त ट्रैकिंग के लिए एक ग्राफ़िकल यूजर इंटरफेस

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