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. איתור של אנדוגני SCAP חלבון בתאים אנושיים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תרבות טיפול בתאים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> זרע ~ 1 × 10 6 תאים U87 בצלחת 10 ס&quot;מ עם בינוני הנשר שונה של Dulbecco (DMEM) בתוספת 5% נסיוב עוברי שור (FBS) ו דגירה התא...

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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>

במחקר זה, אנו מתארים פרוטוקול עבור הבידוד של שברי קרום בתאים אנושיים וכן הם אחראים לעריכה של הדגימות לצורך זיהוי של -glycosylation SCAP N ו- חלבון הכולל באמצעות כתם מערבי. בנוסף, אנו מספקים שיטה לפקח סחר SCAP באמצעות GFP-תיוג מיקרוסקופיה confocal. השיטה משמשת במיוחד כדי לנתח את חל?...

דה-רגולציה של מטבוליזם שומנים הוא מאפיין נפוץ של מחלות הסרטן ומטבוליות 1-6. בתהליכים אלו, חלבונים קושרי אלמנט מווסת sterol (SREBPs), משפחה של גורמי שעתוק, לשחק תפקיד קריטי שליטה על הביטוי של גנים חשובים עבור ספיגת וסינתזה של כולסטרול, חומצות שומן, פוספוליפידים 7-10. SREBPs, כולל-1a SREBP, SREBP-1c, ו SREBP-2, מסונתזים כמו מבשרי פעיל אשר נקלטות על ידי reticulum endoplasmic (ER) קרום מכוח שני תחומים הטרנסממברני 7. N-הסופי של SREBPs מכיל תחום ה- DNA מחייבת עבור transactivation של גני המטרה 11. C- הסופית של מבשרי SREBP נקשר חלבון הפעלת מחשוף SREBP (SCAP) 12,13, חלבון הממברנה polytopic זה ממלא תפקיד חיוני בהסדרת יציבות SREBP והפעלה 14-16. implemenשינוע של פונקציה תעתיק דורש טרנסלוקציה של המתחם SCAP / SREBP מ ER אל Golgi, שבו שני פרוטאזות ברצף לבקע SREBP ולשחרר שבר N- מסוף שלה, אשר לאחר מכן נכנס לתוך הגרעין עבור transactivation של גנים lipogenic 7. בתהליכים אלו, רמות של סטרולים בממברנה ER לשלוט היציאה של המתחם SCAP / SREBP מ ER 7,17. בתנאים sterol גבוהה, sterol נקשר SCAP או ER-תושב-induced אינסולין הגן חלבון-1 (Insig-1) או -2 (Insig-2), שיפור איגוד SCAP עם Insigs כי לשמר את מורכבות SCAP / SREBP ב ER 18-20. כאשר רמות sterol להקטין, SCAP מנתק עם Insigs. זה מוביל לשינוי קונפורמציה SCAP, המאפשר אינטראקציה SCAP עם חלבונים המעיל משותף (COP) מורכב השנייה. המתחם מתווכת שילוב של המתחם SCAP / SREBP לתוך שלפוחית ניצני ומפנה והובלתו מ ER אל Golgi 21,22. עם translocation אל Golgi, SREBPs הם ביקע ברצף ידי אתר-1 ו אתר-2 פרוטאזות, שמוביל את שחרורו של N הסופית- 7,23-29. חלבון SCAP נושאת שלוש N -linked אוליגוסכרידים ב asparagine (N) ממקם N263, N590, N641 ו 15. אנו חשפנו לאחרונה כי בתיווך גלוקוז N -glycosylation של SCAP באתרים אלה הוא תנאי מוקדם לסחר SCAP / SREBP מ ER אל Golgi בתנאי sterol נמוכים 30-32. הפסד של glycosylation SCAP באמצעות מוטציה של כל שלושת asparagine כדי גלוטמין (NNN ל QQQ) משבית את הסחר של המתחם SCAP / SREBP ותוצאות חוסר היציבות של החלבון SCAP והפחתת SREBP הפעלה 31. הנתונים האחרונים שלנו הוכיחו SREBP-1 מופעלת מאוד גליובלסטומה והוא מווסת על ידי SCAP N -glycosylation 4,31,33,34. מיקוד איתות SCAP / SREBP-1 מתגלה כאסטרטגיה חדשה לטיפול בממאירויות ו- s מטבוליתyndromes 1,3,35-38. לכן, חשוב לפתח שיטה יעילה לנתח את חלבון SCAP ו- N -glycosylation הרמות ולפקח הסחר שלה בתאים אנושיים ורקמות חולות. האזור luminal של חלבון SCAP (חומצות אמינו 540-707) בחדר המיון מכיל שני אתרים -glycosylation N (N590 ו- N641) כי מוגנים מפני proteolysis כאשר קרומים שלמים מטופלים עם טריפסין 15. יש שבר לומינל זה משקל מולקולרי של ~ 30 kDa כי הוא קטן מספיק כדי לאפשר את הרזולוציה של וריאנטים glycosylated הפרט של SCAP ידי ג&#39;ל אלקטרופורזה polyacrylamide סולפט dodecyl נתרן (SDS-PAGE) 15,31. כאן, אנו מספקים שיטה לגילוי N -glycosylation של SCAP ואת החלבון הכולל בתאים אנושיים. פרוטוקול זה נגזר השיטה המתוארת בפרסומים ממעבדת בראון וגולדשטיין 15 והפרסום האחרון שלנו 31. הפרוטוקול ניתן להשתמש tהוא ללמוד חלבון SCAP מתאי יונקים.

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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.

איור 1 מציג את זיהוי של חלבון SCAP אנדוגני SREBP-1 טופס גרעין בתאי U87 גליובלסטומה אדם בתגובה לגלוקוזה גירוי באמצעות כתם מערבי. SCAP חלבון הממברנה מזוהה &quot;שבריר קרום&quot;. צורת הגרעין (N-terminal) של SREBP-1 מזוהית &quot;תמציות גרעיניות&quot;. רמת חלבון SCAP (PDI מש...

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

אנו מתארים שיטה שונה לבידוד שבריר קרום מתאי אדם הכנת מדגם לצורך זיהוי של -glycosylation SCAP N ו- חלבון הכולל באמצעות כתם מערבי. בנוסף, אנו מציגים שיטה GFP תיוג לפקח סחר SCAP באמצעות מיקרוסקופ confocal. פרוטוקול זה יכול לשמש במעבדות ביולוגיה קבועות.

ניתוח של SCAP<em&gt; N</em&gt; -glycosylation וסחר בבני אדם תאים

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

וידאו ביואינפורמטיקה ניתוח גדילה אנושי בתאי גזע עובריים קולוני

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

ניתוח בידוד פנוטיפי של Murine בתאי גזע hematopoietic ו-Lineage מחויבים אבות

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

ניתוח RNA-seq של Transcriptomes בטרומבין טופל ותאי שליטה אנושי ריאת כלי דם האנדותל

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

אינדוקציה וניתוח של אפיתל לmesenchymal מעבר

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

תאי גזע יעיל דור מושרה אדם פלוריפוטנטיים מתאים הסומטיים של אדם עם סנדאי וירוס

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

אפיון תאי גזע עוברי האנושי על ידי הפרט כמותי 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

מעקב אחר שינויים בהשפעת סמים בסחר פוסט-הפנמת קולטן על ידי ניתוח 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

זמן לשגות הדמיה ברזולוציה גבוהה וניתוח אוטומטי של Dynamics microtubule In Living האדם טבורי וריד לתאי אנדותל

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

הדמיה של תא חי ו 3D הניתוח של אנגיוטנסין קולטן סוג סחר 1a בתאי הכליה טרנספקציה אדם העוברי באמצעות מיקרוסקופ Confocal

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

ממשק משתמש גרפי למעקב בסיוע תוכנה של ריכוז חלבון בהפרעות ניידות דינמיות

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