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.

ヒト細胞における内因性SCAPタンパク質の1検出<ol><li>細胞培養および治療<ol><li>ダルベッコ改変イーグル培地（DMEM）を用いて10cmディッシュでシード〜1×10 6個のU87細胞を、5％ウシ胎児血清（FBS）を補充し、処理前に24時間37℃で細胞を、5％CO 2でインキュベートします。 </li><li>リン酸緩衝生理食塩水（PBS）で細胞を1回洗浄し、その後12時間（5ミリモル）を伴うまたはグルコー?...

<ol>
	<li>Ru, P., Williams, T. M., Chakravarti, A., 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=Tumor+metabolism+of+malignant+gliomas.">Tumor metabolism of malignant gliomas.</a> Cancers (Basel). 5, 1469-1484 (2013).</li><li>Moon, Y. A., 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=The+Scap/SREBP+pathway+is+essential+for+developing+diabetic+fatty+liver+and+carbohydrate-induced+hypertriglyceridemia+in+animals.">The Scap/SREBP pathway is essential for developing diabetic fatty liver and carbohydrate-induced hypertriglyceridemia in animals.</a> Cell metabolism. 15, 240-246 (2012).</li><li>Guo, D., Bell, E. H., 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=Lipid+metabolism+emerges+as+a+promising+target+for+malignant+glioma+therapy.">Lipid metabolism emerges as a promising target for malignant glioma therapy.</a> CNS Oncology. 2, 289-299 (2013).</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=The+AMPK+agonist+AICAR+inhibits+the+growth+of+EGFRvIII-expressing+glioblastomas+by+inhibiting+lipogenesis.">The AMPK agonist AICAR inhibits the growth of EGFRvIII-expressing glioblastomas by inhibiting lipogenesis.</a> Proceedings of the National Academy of Sciences of the United States of America. , 12932-12937 (2009).</li><li>Menendez, J. A., Lupu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid+synthase+and+the+lipogenic+phenotype+in+cancer+pathogenesis.">Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis.</a> Nat Rev Cancer. 7, 763-777 (2007).</li><li>Guo, D., Cloughesy, T. F., Radu, C. G., Mischel, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPK:+A+metabolic+checkpoint+that+regulates+the+growth+of+EGFR+activated+glioblastomas.">AMPK: A metabolic checkpoint that regulates the growth of EGFR activated glioblastomas.</a> Cell Cycle. 9, 211-212 (2010).</li><li>Goldstein, J. L., DeBose-Boyd, R. A., Brown, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+sensors+for+membrane+sterols.">Protein sensors for membrane sterols.</a> Cell. 124, 35-46 (2006).</li><li>Shao, W., Espenshade, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+Roles+for+SREBP+in+Metabolism.">Expanding Roles for SREBP in Metabolism.</a> Cell metabolism. 16, 414-419 (2012).</li><li>Nohturfft, A., Zhang, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+of+lipid+metabolism+in+membrane+biogenesis.">Coordination of lipid metabolism in membrane biogenesis.</a> Annual review of cell and developmental biology. 25, 539-566 (2009).</li><li>Jeon, T. I., Osborne, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SREBPs:+metabolic+integrators+in+physiology+and+metabolism.">SREBPs: metabolic integrators in physiology and metabolism.</a> Trends in endocrinology and metabolism: TEM. 23, 65-72 (2012).</li><li>Sato, R., 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=Assignment+of+the+membrane+attachment,+DNA+binding,+and+transcriptional+activation+domains+of+sterol+regulatory+element-binding+protein-1+(SREBP-1).">Assignment of the membrane attachment, DNA binding, and transcriptional activation domains of sterol regulatory element-binding protein-1 (SREBP-1).</a> The Journal of biological chemistry. 269, 17267-17273 (1994).</li><li>Sakai, J., Nohturfft, A., Goldstein, J. L., Brown, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+sterol+regulatory+element-binding+proteins+(SREBPs)+at+site-1+requires+interaction+with+SREBP+cleavage-activating+protein.+Evidence+from+in+vivo+competition+studies.">Cleavage of sterol regulatory element-binding proteins (SREBPs) at site-1 requires interaction with SREBP cleavage-activating protein. Evidence from in vivo competition studies.</a> The Journal of biological chemistry. 273, 5785-5793 (1998).</li><li>Sakai, J., 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=Identification+of+complexes+between+the+COOH-terminal+domains+of+sterol+regulatory+element-binding+proteins+(SREBPs)+and+SREBP+cleavage-activating+protein.">Identification of complexes between the COOH-terminal domains of sterol regulatory element-binding proteins (SREBPs) and SREBP cleavage-activating protein.</a> The Journal of biological chemistry. 272, 20213-20221 (1997).</li><li>Hua, X., Nohturfft, A., Goldstein, J. L., Brown, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sterol+resistance+in+CHO+cells+traced+to+point+mutation+in+SREBP+cleavage-activating+protein.">Sterol resistance in CHO cells traced to point mutation in SREBP cleavage-activating protein.</a> Cell. 87, 415-426 (1996).</li><li>Nohturfft, A., Brown, M. S., Goldstein, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topology+of+SREBP+cleavage-activating+protein,+a+polytopic+membrane+protein+with+a+sterol-sensing+domain.">Topology of SREBP cleavage-activating protein, a polytopic membrane protein with a sterol-sensing domain.</a> The Journal of biological chemistry. 273, 17243-17250 (1998).</li><li>Nohturfft, A., Hua, X., Brown, M. S., Goldstein, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+G-to-A+substitution+in+a+single+codon+of+SREBP+cleavage-activating+protein+causes+sterol+resistance+in+three+mutant+Chinese+hamster+ovary+cell+lines.">Recurrent G-to-A substitution in a single codon of SREBP cleavage-activating protein causes sterol resistance in three mutant Chinese hamster ovary cell lines.</a> Proc Natl Acad Sci U S A. 93, 13709-13714 (1996).</li><li>Espenshade, P. J., Hughes, A. L. <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+sterol+synthesis+in+eukaryotes.">Regulation of sterol synthesis in eukaryotes.</a> Annu Rev Genet. 41, 401-427 (2007).</li><li>Yang, 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=Crucial+step+in+cholesterol+homeostasis:+sterols+promote+binding+of+SCAP+to+INSIG-1,+a+membrane+protein+that+facilitates+retention+of+SREBPs+in+ER.">Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER.</a> Cell. 110, 489-500 (2002).</li><li>Yabe, D., Brown, M. S., Goldstein, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insig-2,+a+second+endoplasmic+reticulum+protein+that+binds+SCAP+and+blocks+export+of+sterol+regulatory+element-binding+proteins.">Insig-2, a second endoplasmic reticulum protein that binds SCAP and blocks export of sterol regulatory element-binding proteins.</a> Proc Natl Acad Sci U S A. 99, 12753-12758 (2002).</li><li>Yabe, D., Xia, Z. P., Adams, C. M., Rawson, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+mutations+in+sterol-sensing+domain+of+SCAP+block+interaction+with+insig+and+render+SREBP+cleavage+insensitive+to+sterols.">Three mutations in sterol-sensing domain of SCAP block interaction with insig and render SREBP cleavage insensitive to sterols.</a> Proc Natl Acad Sci U S A. 99, 16672-16677 (2002).</li><li>Espenshade, P. J., Li, W. P., Yabe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sterols+block+binding+of+COPII+proteins+to+SCAP,+thereby+controlling+SCAP+sorting+in+ER.">Sterols block binding of COPII proteins to SCAP, thereby controlling SCAP sorting in ER.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 11694-11699 (2002).</li><li>Nohturfft, A., Yabe, D., Goldstein, J. L., Brown, M. S., Espenshade, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+step+in+cholesterol+feedback+localized+to+budding+of+SCAP+from+ER+membranes.">Regulated step in cholesterol feedback localized to budding of SCAP from ER membranes.</a> Cell. 102, 315-323 (2000).</li><li>Sakai, J., 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=Sterol-regulated+release+of+SREBP-2+from+cell+membranes+requires+two+sequential+cleavages,+one+within+a+transmembrane+segment.">Sterol-regulated release of SREBP-2 from cell membranes requires two sequential cleavages, one within a transmembrane segment.</a> Cell. 85, 1037-1046 (1996).</li><li>Wang, X., Sato, R., Brown, M. S., Hua, X., Goldstein, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SREBP-1,+a+membrane-bound+transcription+factor+released+by+sterol-regulated+proteolysis.">SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis.</a> Cell. 77, 53-62 (1994).</li><li>Brown, M. S., Goldstein, J. 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+proteolytic+pathway+that+controls+the+cholesterol+content+of+membranes,+cells,+and+blood.">A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood.</a> Proceedings of the National Academy of Sciences of the United States of America. 96, 11041-11048 (1999).</li><li>Rawson, R. B., DeBose-Boyd, R., Goldstein, J. L., Brown, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Failure+to+cleave+sterol+regulatory+element-binding+proteins+(SREBPs)+causes+cholesterol+auxotrophy+in+Chinese+hamster+ovary+cells+with+genetic+absence+of+SREBP+cleavage-activating+protein.">Failure to cleave sterol regulatory element-binding proteins (SREBPs) causes cholesterol auxotrophy in Chinese hamster ovary cells with genetic absence of SREBP cleavage-activating protein.</a> J Biol Chem. 274, 28549-28556 (1999).</li><li>Zelenski, N. G., Rawson, R. B., Brown, M. S., Goldstein, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+topology+of+S2P,+a+protein+required+for+intramembranous+cleavage+of+sterol+regulatory+element-binding+proteins.">Membrane topology of S2P, a protein required for intramembranous cleavage of sterol regulatory element-binding proteins.</a> J Biol Chem. 274, 21973-21980 (1999).</li><li>Goldstein, J. L., Brown, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Century+of+Cholesterol+and+Coronaries:+From+Plaques+to+Genes+to+Statins.">A Century of Cholesterol and Coronaries: From Plaques to Genes to Statins.</a> Cell. 161, 161-172 (2015).</li><li>Espenshade, P. J., Cheng, D., Goldstein, J. L., Brown, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocatalytic+processing+of+site-1+protease+removes+propeptide+and+permits+cleavage+of+sterol+regulatory+element-binding+proteins.">Autocatalytic processing of site-1 protease removes propeptide and permits cleavage of sterol regulatory element-binding proteins.</a> The Journal of biological chemistry. 274, 22795-22804 (1999).</li><li>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=SCAP+links+glucose+to+lipid+metabolism+in+cancer+cells.">SCAP links glucose to lipid metabolism in cancer cells.</a> Mol Cell Oncol. 3, (2016).</li><li>Cheng, C., 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=Glucose-Mediated+N-glycosylation+of+SCAP+Is+Essential+for+SREBP-1+Activation+and+Tumor+Growth.">Glucose-Mediated N-glycosylation of SCAP Is Essential for SREBP-1 Activation and Tumor Growth.</a> Cancer Cell. 28, 569-581 (2015).</li><li>Shao, W., Espenshade, P. 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>

本研究では、ヒト細胞における膜画分の単離およびウエスタンブロットを用いてSCAP N -glycosylationおよび総タンパク質の検出のためのサンプルを調製するためのプロトコルを説明します。我々は、さらに、GFP標識し、共焦点顕微鏡を用いてSCAP輸送を監視するための方法を提供します。この方法は、特に膜タンパク質を分析するために使用され、SCAP Nの -glycosylationおよび輸送を研究...

脂質代謝の規制緩和は、癌および代謝性疾患1-6の一般的な特徴です。これらの方法では、ステロール調節エレメント結合タンパク質（SREBPs）、転写因子のファミリーは、コレステロールの取り込みおよび合成のための重要な遺伝子の発現の制御に重要な役割を果たし、脂肪酸、及びリン脂質7-10。 SREBP-1aを、SREBP-1C、およびSREBP-2を含むSREBPsは、2つの膜貫通ドメイン7によって小胞体（ER）膜に結合し不活性前駆体として合成されます。 SREBPsのN末端は、標的遺伝子11のトランスのためのDNA結合ドメインが含まれています。 SREBP前駆体のC末端は、SREBP切断活性化タンパク質（SCAP）12,13、SREBPの安定性及び活性化14-16の調節において重要な役割を果たしているポリトピック膜タンパク質に結合します。 implemen転写機能のテーションは、2つのプロテアーゼが順次SREBPを切断した後、脂質生成遺伝子7のトランスのための核に入り、そのN末端断片を、解放ゴルジへの小胞体からSCAP / SREBP複合体の転座を必要とします。これらのプロセスでは、ER膜中のステロールのレベルは、ER 7,17からSCAP / SREBP複合体の出口を制御します。高ステロール条件下では、ステロールはでSCAP / SREBP複合体を保持InsigsとSCAPの関連性を高め、SCAPまたはERに存在するインスリン誘導遺伝子タンパク質-1（Insig-1）または-2（Insig-2）に特異的に結合しますER 18-20。ステロールのレベルが減少すると、SCAPはInsigsで解離します。これは一般的なコートタンパク質（COP）II複合体とSCAPの相互作用を可能にするSCAPコンホメーション変化をもたらします。複合体は、出芽小胞にSCAP / SREBP複合体の取り込みを仲介し、ゴルジ体21,22にERからの輸送を指示します。 transloc時にゴルジ体へエーション、SREBPsを順次N末端7,23-29の放出をもたらす、サイト1とサイト-2プロテアーゼによって切断されています。 SCAPタンパク質は、Nはアスパラギンでオリゴ糖を-結合3運ぶ（N）は、N263、N590、N641および15を位置決めします 。我々は最近、これらのサイトにおけるSCAPのグルコース媒介Nの -glycosylationが低いステロール条件30-32の下でERからゴルジへのSCAP / SREBP輸送のための必須条件であることを明らかにしました。 （QQQへNNN）グルタミンにすべての3つのアスパラギンの変異を介したSCAPグリコシル化の損失は、SCAPタンパク質とSREBPの活性化31の減少が不安定でSCAP / SREBP複合体と結果の人身売買を無効にします。私たちの最近のデータはまた、SREBP-1は非常に神経膠芽腫で活性化し、SCAPのN -glycosylation 4,31,33,34によって規制されることを示します。 SCAP / SREBP-1シグナル伝達が悪性腫瘍を治療するための新たな戦略および代謝秒として浮上しているターゲティングyndromes 1,3,35-38。したがって、SCAPタンパク質およびN -glycosylationレベルを分析し、ヒト細胞と患者組織における輸送を監視する有効な方法を開発することが重要です。 ERにおけるSCAPタンパク質（アミノ酸540から707）の管腔領域は、無傷の膜をトリプシン15で処理した場合にタンパク質分解から保護された2つのN -glycosylationサイト（N590およびN641）が含まれています。この管腔フラグメントは、ドデシル硫酸ナトリウムポリアクリルアミドゲル電気泳動（SDS-PAGE）15,31によってSCAPの個々のグリコシル化バリアントの解決を可能にするのに十分に小さい〜30kDaの分子量を有します。ここでは、N個の SCAPの-glycosylationおよびヒト細胞中の全タンパク質を検出するための方法を提供します。このプロトコルはブラウンとゴールドスタイン実験室15と我々の最近の刊行物31からの刊行物に記載された方法に由来します。プロトコルは、Tで使用することができ彼は、哺乳動物細胞からSCAPタンパク質の研究します。

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

図1は、ウエスタンブロットを使用して、グルコース刺激に応答したヒト神経膠芽腫U87細胞における内因性SCAPタンパク質およびSREBP-1の核形態の検出を示します。膜タンパク質のSCAPを「膜画分」で検出されます。 SREBP-1の核型（N末端）は、「核抽出物」で検出されます。 SCAPタンパク質のレベルは顕著にグルコース刺激によって増強され、SREBP-1の核形...

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

我々は、ヒト細胞およびウェスタンブロットを用いてSCAP N -glycosylationおよび総タンパク質の検出のためのサンプル調製物からの膜画分を単離するための改良法を記載します。我々はさらに、共焦点顕微鏡を用いてSCAPの人身売買を監視するために、GFP-標識法をご紹介します。このプロトコルは、通常の生物学実験室で使用することができます。

SCAPの分析<em&gt; N</em&gt;ヒト細胞における-glycosylationと人身売買

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

ヒト胚性幹細胞のコロニーの成長のビデオバイオインフォマティクス解析

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

マウス造血幹細胞およびリネージュコミット前駆細胞の表現型の解析と分離

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のトロンビン処理におけるトランスクリプトームの解析と制御ヒト肺微小血管内皮細胞

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

間葉転換を誘導し、上皮の分析

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

仙台ウイルスのヒト体細胞からの効率的な次世代ヒューマン誘導多能性幹細胞

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

定量的RT-PCRによって個々のヒト胚性幹細胞のプロファイリング

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解析による受容体後の内在トラフィッキングにおける薬物誘発性の変更の追跡

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

リビングヒト臍帯静脈内皮細胞における高解像度タイムラプスイメージングと微小管ダイナミクスの自動解析

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

共焦点顕微鏡を使用してトランスフェクトされたヒト胚性腎細胞におけるアンジオテンシン受容体1a型の人身売買のライブセルイメージングと3D解析

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

動的細胞突起におけるソフトウェア支援トラッキングのためのグラフィカルユーザインタフェース

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