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. Обнаружение эндогенного СПДК белка в клетках человека <ol><li> Культура клеток и лечение <ol><li> Семенной ~ 1 × 10 6 U87 клеток в 10 см чашку с Дульбекко в модификации Дульбекко (DMEM) с добавлением 5% фетальной бычьей сыворотки (FBS) и инкубируют клетки при 37 ° С и 5% СО 2 в течение 24 ч перед о...

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	<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>

В этом исследовании мы опишем протокол для выделения мембранных фракций в клетках человека и для приготовления образцов для обнаружения СПДК N -glycosylation и общего белка с использованием вестерн - блот. Кроме того, мы предоставить способ для контроля за оборотом SCAP с помощью GFP мечение...

Нарушение регуляции липидного обмена является общей характеристикой рака и болезней обмена веществ , 1-6. В этих процессах, стеринов регуляторный элемент-связывающих белков (SREBPs), семейство факторов транскрипции, играют решающую роль в контроле экспрессии генов , имеющих важное значение для поглощения и синтеза холестерина, жирных кислот, фосфолипидов и 7-10. SREBPs, в том числе SREBP-1a, SREBP-1c и SREBP-2, синтезируются в виде неактивных предшественников , которые связываются с мембраной эндоплазматического ретикулума (ER) в силу двух трансмембранных доменов 7. N-конец SREBPs содержит ДНК-связывающий домен трансактивации генов - мишеней 11. С-конец SREBP предшественников связывается с SREBP скола-активирующий белок (SCAP) 12,13, в политопная мембранный белок , который играет ключевую роль в регуляции стабильности SREBP и активации 14-16. implemenставление транскрипционной функции требует транслокацию СПДК / SREBP комплекса из ЭР к Гольджи, где два протеаз последовательно расщеплять SREBP и освободить его N-концевой фрагмент, который затем входит в ядро для трансактивации генов липогенных 7. В этих процессах, уровни стеринов в ЭР мембраны контролируют выход СПДК / SREBP комплекса из ЭР 7,17. При высоких условиях стеролов, стеринов связывается с СПДК или ER-резидент инсулин-индуцированный ген белка-1 (1-экс порте) или -2 (2-экс порте), усиливая ассоциацию с СПДК Insigs, сохраняющих СПДК / SREBP комплекс в ER 18-20. Когда уровни стеролов уменьшают, СПДК диссоциирует с Insigs. Это приводит к конформационным изменениям SCAP, что позволяет СПДК взаимодействие с общими покрывающих белков комплекса (КС) II. Комплекс является медиатором включение СПДК / SREBP комплекса в многообещающий везикул и направляет его транспортировку из ЭР к Гольджи 21,22. После Translocвания к Гольджи, SREBPs последовательно расщепляется Зону-1 и Site-2 протеаз, что приводит к выделению из N-конец 7,23-29. СПДК белок несет три N -связанной олигосахариды на аспарагин (N) позиции N263, N590 и N641 15. Недавно мы показали , что глюкоза-опосредованного N -glycosylation из СПДК в этих местах является необходимым условием условием для торговли СПДК / SREBP из ЭР к Гольджи в условиях низких стеролов 30-32. Потеря SCAP гликозилирования с помощью мутации всех трех аспарагина на глутамин (NNN в QQQ) отключает торговлю СПДК SREBP комплекса и результаты / в нестабильности SCAP белка и уменьшения активации SREBP 31. Наши последние данные показывают также , что SREBP-1 сильно активируется в глиобластомы и регулируется SCAP N -glycosylation 4,31,33,34. Ориентация СПДК / SREBP-1 сигнализации появляется в качестве новой стратегии для лечения злокачественных новообразований и метаболических Syndromes 1,3,35-38. Поэтому важно разработать эффективный метод для анализа уровней СПДК белка и N -glycosylation и контроля за его оборотом человеческих клеток и тканей пациента. Полостную область СПДК белка (аминокислоты 540-707) в ER содержит два участка -glycosylation N (N590 и N641), которые защищены от протеолиза при неповрежденных мембран обрабатывают трипсином 15. Этот фрагмент полостную имеет молекулярную массу ~ 30 кДа , который достаточно мал , чтобы позволить разрешение отдельных гликозилированных вариантов СПДК методом электрофореза в полиакриламидном геле в сульфат натрия додецилсульфата (SDS-PAGE) 15,31. Здесь мы предлагаем способ для обнаружения N -glycosylation из СПДК и общего белка в клетках человека. Этот протокол является производным от метода , описанного в публикации из лаборатории Брауна и Goldstein 15 и нашей недавней публикации 31. Протокол может быть использован в тон изучение SCAP белка из клеток млекопитающих.

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			<td height="21" style="height:21px;width:259px;">X-treme GENE HP DNA Transfection Reagent&#160;</td>
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			<td>Life Technologies</td>
			<td>31985-070</td>
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			<td height="21" style="height:21px;">trypsin&#160;</td>
			<td>Sigma</td>
			<td>T6567</td>
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			<td>P7367</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Anti-SCAP (9D5) antibody&#160;&#160;</td>
			<td>Santa Cruz</td>
			<td>sc-13553</td>
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			<td height="21" style="height:21px;">GFP antibody&#160;</td>
			<td>Roche</td>
			<td>11814460001</td>
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			<td height="21" style="height:21px;">SREBP-1 antibody (IgG-2A4)</td>
			<td>BD Pharmingen</td>
			<td>557036</td>
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			<td height="21" style="height:21px;">PDI Antibody (H-17)</td>
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			<td height="21" style="height:21px;">SCAP antibody (a.a 450-500)</td>
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			<td>A303-554A</td>
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			<td height="21" style="height:21px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) without glucose, pyruvate and glutamine&#160;&#160;</td>
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			<td>17-207-CV</td>
			<td>Add 1 Mm Pyravate and 4 mM Glutamine before use</td>
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			<td height="21" style="height:21px;">leupeptin</td>
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			<td>L2884</td>
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			<td height="21" style="height:21px;">PMSF</td>
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			<td>P7626</td>
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			<td height="21" style="height:21px;">DTT</td>
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			<td height="21" style="height:21px;">ALLN</td>
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			<td>A6185</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Nitrocellulose membrane</td>
			<td>GE</td>
			<td>RPN3032D</td>
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			<td height="21" style="height:21px;">EDTA Solution 0.5 M PH8.5 100 ml&#160;</td>
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			<td height="21" style="height:21px;">EGTA 0.5 M sterile (PH 8.0) 50 ml</td>
			<td>Fisher Scentific</td>
			<td>50255956</td>
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			<td height="21" style="height:21px;">HyClone FBS</td>
			<td>Thermo scientific</td>
			<td>SH3007103</td>
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			<td height="21" style="height:21px;">glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
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		<tr height="21">
			<td height="21" style="height:21px;">&#946;-mercaptoethanol&#160;</td>
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			<td>M3148</td>
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			<td height="21" style="height:21px;">bromophenol blue</td>
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			<td>B8026</td>
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			<td height="21" style="height:21px;">prolong gold antifade reagent with dapi</td>
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			<td>P36935</td>
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			<td height="21" style="height:21px;">L-glutamine (200 mM)</td>
			<td>life technologies</td>
			<td>25030081</td>
			<td></td>
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			<td height="21" style="height:21px;">sodium pyruvate</td>
			<td>life technologies</td>
			<td>11360-070</td>
			<td></td>
		</tr>
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analysis of scap n-glycosylation and trafficking in human cells

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

На рисунке 1 показано обнаружение эндогенного белка СПДК и SREBP-1 ядерной формы в клетках глиобластомы U87 человека в ответ на глюкозу стимуляции с помощью вестерн - блот. Мембранный белок СПДК обнаруживается в &quot;мембранной фракции&quot;. Форма ядра (N-концевой) ...

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

Описан модифицированный метод выделения мембранной фракции из клеток человека и подготовки проб для обнаружения СПДК N -glycosylation и общего белка с использованием вестерн - блот. Введем еще метод GFP-маркировки наблюдать за торговлей СПДК использованием конфокальной микроскопии. Этот протокол может быть использован в обычных биологических лабораториях.

Анализ СПДК<em&gt; N</em&gt; -glycosylation И торговле человеческими клетками

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

Видео биоинформатики Анализ человеческих эмбриональных роста клеток колонии стволовых

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

Фенотипический анализ и выделение мышей гемопоэтических стволовых клеток и Lineage, совершенных прародителей

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

РНК-след Анализ Transcriptomes в тромбин обращению и контролю человека легочной микрососудистой эндотелиальных клеток

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

Индукционные и анализ эпителиальных к мезенхимальных перехода

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

Эффективная генерация человека индуцированных плюрипотентных стволовых клеток из человеческих соматических клеток с Сендай-вируса

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

Профилирования Индивидуальный человеческих эмбриональных стволовых клеток путем количественного 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

Отслеживание наркотиков изменений, вызванных в рецептора после интернализации торговле от анализа Colocalizational

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

Высокое разрешение Покадровый изображений и автоматизированный анализ динамики микротрубочек в живых человеческих эндотелиальных клеток пупочной вены

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

Живая съемка Cell и 3D Анализ ангиотензина типа 1a Торговля в трансфицированных человеческих эмбриональных клеток почки с помощью конфокальной микроскопии

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

Графический пользовательский интерфейс для отслеживания концентрации белка в программном сопровождении в динамических клеточных выпячиваниях

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