Name: quantitative measurement of &#947;-secretase-mediated amyloid precursor protein and notch cleavage in cell-based luciferase reporter assay platforms
Uploaded: 2018-01-25T12:30:00
Description: Watch this Scientific Journal Video about Quantitative Measurement of ÃŽÂ³-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms at JoVE.com

Forfatterne takke Core facilitet af Institut for Cellulær og organismens biologi, Academia Sinica, for teknisk support. Denne undersøgelse blev støttet af Ministeriet for videnskab og teknologi, Taiwan (mest 103-2320-B-001-016-MY3 til Y.-F. L.), programmet for Translationel Innovation af biofarmaceutiske udvikling – teknologi støtte Platform akse ordning (NP7 til Y.-F.L.), og Academia Sinica (til Y.-F.L.).

1. måling af Luciferase Reporter signaler, som svarer til γ-Secretase spaltning af APP-C99 eller NΔE
Bemærk: Der henvises til tidligere publikationer for detaljerede beskrivelser af generation af CG og NG celler20,21.
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Seed NG eller CG celler (20, 000 celler/brønd) på 96-brønd mikroplader på en endelige mængden af 200 μl/brønd i vækstmedium, der er sammensat af Dulbeccos modificerede Eagle Medium (DMEM) pl...

<ol>
	<li>Selkoe, D. J., Hardy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+hypothesis+of+Alzheimer's+disease+at+25+years.">The amyloid hypothesis of Alzheimer's disease at 25 years.</a> EMBO Mol Med. 8 (6), 595-608 (2016).</li><li>Wolfe, M. S., Guenette, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=APP+at+a+glance.">APP at a glance.</a> J Cell Sci. 120 (Pt 18), 3157-3161 (2007).</li><li>Karran, E., Mercken, M., De Strooper, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+cascade+hypothesis+for+Alzheimer's+disease:+an+appraisal+for+the+development+of+therapeutics.">The amyloid cascade hypothesis for Alzheimer's disease: an appraisal for the development of therapeutics.</a> Nat Rev Drug Discov. 10 (9), 698-712 (2011).</li><li>Citron, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease:+strategies+for+disease+modification.">Alzheimer's disease: strategies for disease modification.</a> Nat Rev Drug Discov. 9 (5), 387-398 (2010).</li><li>Vassar, R., Kovacs, D. M., Yan, R., Wong, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beta-secretase+enzyme+BACE+in+health+and+Alzheimer's+disease:+regulation,+cell+biology,+function,+and+therapeutic+potential.">The beta-secretase enzyme BACE in health and Alzheimer's disease: regulation, cell biology, function, and therapeutic potential.</a> J Neurosci. 29 (41), 12787-12794 (2009).</li><li>Parks, A. L., Curtis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presenilin+diversifies+its+portfolio.">Presenilin diversifies its portfolio.</a> Trends Genet. 23 (3), 140-150 (2007).</li><li>Kukar, T. L., 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=Substrate-targeting+gamma-secretase+modulators.">Substrate-targeting gamma-secretase modulators.</a> Nature. 453 (7197), 925-929 (2008).</li><li>Kounnas, M. Z., 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=Modulation+of+gamma-secretase+reduces+beta-amyloid+deposition+in+a+transgenic+mouse+model+of+Alzheimer's+disease.">Modulation of gamma-secretase reduces beta-amyloid deposition in a transgenic mouse model of Alzheimer's disease.</a> Neuron. 67 (5), 769-780 (2010).</li><li>Thathiah, 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+orphan+G+protein-coupled+receptor+3+modulates+amyloid-beta+peptide+generation+in+neurons.">The orphan G protein-coupled receptor 3 modulates amyloid-beta peptide generation in neurons.</a> Science. 323 (5916), 946-951 (2009).</li><li>Flajolet, M., 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=Regulation+of+Alzheimer's+disease+amyloid-beta+formation+by+casein+kinase+I.">Regulation of Alzheimer's disease amyloid-beta formation by casein kinase I.</a> Proc Natl Acad Sci U S A. 104 (10), 4159-4164 (2007).</li><li>Eisele, Y. S., 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=Gleevec+increases+levels+of+the+amyloid+precursor+protein+intracellular+domain+and+of+the+amyloid-beta+degrading+enzyme+neprilysin.">Gleevec increases levels of the amyloid precursor protein intracellular domain and of the amyloid-beta degrading enzyme neprilysin.</a> Mol Biol Cell. 18 (9), 3591-3600 (2007).</li><li>He, G., 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=Gamma-secretase+activating+protein+is+a+therapeutic+target+for+Alzheimer's+disease.">Gamma-secretase activating protein is a therapeutic target for Alzheimer's disease.</a> Nature. 467 (7311), 95-98 (2010).</li><li>Haapasalo, A., Kovacs, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+substrates+of+presenilin/gamma-secretase.">The many substrates of presenilin/gamma-secretase.</a> J Alzheimers Dis. 25 (1), 3-28 (2011).</li><li>Gillman, K. W., 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=Discovery+and+Evaluation+of+BMS-708163,+a+Potent,+Selective+and+Orally+Bioavailable+gamma-Secretase+Inhibitor.">Discovery and Evaluation of BMS-708163, a Potent, Selective and Orally Bioavailable gamma-Secretase Inhibitor.</a> ACS Med Chem Lett. 1 (3), 120-124 (2010).</li><li>Hopkins, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACS+chemical+neuroscience+molecule+spotlight+on+Begacestat+(GSI-953).">ACS chemical neuroscience molecule spotlight on Begacestat (GSI-953).</a> ACS Chem Neurosci. 3 (1), 3-4 (2012).</li><li>Yang, T., Arslanova, D., Gu, Y., Augelli-Szafran, C., Xia, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+gamma-secretase+modulation+differentiates+inhibitor+compound+selectivity+between+two+substrates+Notch+and+amyloid+precursor+protein.">Quantification of gamma-secretase modulation differentiates inhibitor compound selectivity between two substrates Notch and amyloid precursor protein.</a> Mol Brain. 1, 15 (2008).</li><li>McKee, T. D., Loureiro, R. M., Dumin, J. A., Zarayskiy, V., Tate, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+cell-based+method+for+determining+the+gamma-secretase+enzyme+activity+against+both+Notch+and+APP+substrates.">An improved cell-based method for determining the gamma-secretase enzyme activity against both Notch and APP substrates.</a> J Neurosci Methods. 213 (1), 14-21 (2013).</li><li>Basi, G. S., 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=Amyloid+precursor+protein+selective+gamma-secretase+inhibitors+for+treatment+of+Alzheimer's+disease.">Amyloid precursor protein selective gamma-secretase inhibitors for treatment of Alzheimer's disease.</a> Alzheimers Res Ther. 2 (6), 36 (2010).</li><li>Liao, Y. F., Tang, Y. C., Chang, M. Y., Wang, B. J., Hu, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+small+molecular+(D)-leucinamides+as+potent,+Notch-sparing+gamma-secretase+modulators.">Discovery of small molecular (D)-leucinamides as potent, Notch-sparing gamma-secretase modulators.</a> Eur J Med Chem. 79, 143-151 (2014).</li><li>Liao, Y. F., Wang, B. J., Cheng, H. T., Kuo, L. H., Wolfe, 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=Tumor+necrosis+factor-alpha,+interleukin-1beta,+and+interferon-gamma+stimulate+gamma-secretase-mediated+cleavage+of+amyloid+precursor+protein+through+a+JNK-dependent+MAPK+pathway.">Tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma stimulate gamma-secretase-mediated cleavage of amyloid precursor protein through a JNK-dependent MAPK pathway.</a> J Biol Chem. 279 (47), 49523-49532 (2004).</li><li>Wang, B. 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=ErbB2+regulates+autophagic+flux+to+modulate+the+proteostasis+of+APP-CTFs+in+Alzheimer's+disease.">ErbB2 regulates autophagic flux to modulate the proteostasis of APP-CTFs in Alzheimer's disease.</a> Proc Natl Acad Sci U S A. 114 (15), E3129-E3138 (2017).</li><li>Sevigny, 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=The+antibody+aducanumab+reduces+Abeta+plaques+in+Alzheimer's+disease.">The antibody aducanumab reduces Abeta plaques in Alzheimer's disease.</a> Nature. 537 (7618), 50-56 (2016).</li><li>Golde, T. E., Koo, E. H., Felsenstein, K. M., Osborne, B. A., Miele, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gamma-Secretase+inhibitors+and+modulators.">gamma-Secretase inhibitors and modulators.</a> Biochim Biophys Acta. 1828 (12), 2898-2907 (2013).</li><li>Coric, V., 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=Targeting+Prodromal+Alzheimer+Disease+With+Avagacestat:+A+Randomized+Clinical+Trial.">Targeting Prodromal Alzheimer Disease With Avagacestat: A Randomized Clinical Trial.</a> JAMA Neurol. 72 (11), 1324-1333 (2015).</li><li>Mitani, Y., 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=Differential+effects+between+gamma-secretase+inhibitors+and+modulators+on+cognitive+function+in+amyloid+precursor+protein-transgenic+and+nontransgenic+mice.">Differential effects between gamma-secretase inhibitors and modulators on cognitive function in amyloid precursor protein-transgenic and nontransgenic mice.</a> J Neurosci. 32 (6), 2037-2050 (2012).</li><li>Penninkilampi, R., Brothers, H. M., Eslick, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+Agents+Targeting+gamma-Secretase+Increase+Risk+of+Cancer+and+Cognitive+Decline+in+Alzheimer's+Disease+Patients:+A+Systematic+Review+and+Meta-Analysis.">Pharmacological Agents Targeting gamma-Secretase Increase Risk of Cancer and Cognitive Decline in Alzheimer's Disease Patients: A Systematic Review and Meta-Analysis.</a> J Alzheimers Dis. 53 (4), 1395-1404 (2016).</li></ol>

De lovende resultater fra en fase 1b studie af aducanumab immunterapi for annonce har fast etableret Aβ kritiske rolle i patogenesen af annonce22 og foreslår, at anti-Aβ tilgange er stadig en levedygtig strategi for udvikling af anti-annonce narkotika. Her beskriver vi cellebaserede assays til kvantificering af γ-secretase-katalyseret proteolyse af APP-C99 og NΔE ved hjælp af firefly luciferase reporter systemer. APP-C99 og NΔE er de to mest velundersøgte γ-secretase substrater og repræs...

Oligomere former for amyloid-β (Aβ) menes at være den primære årsag til neurodegeneration i hjernen hos patienter med Alzheimers sygdom (AD)1. Aβ peptider er produceret af trinvis kløvningen af amyloid forløber protein (APP), først ved β-secretase og derefter γ-secretase2. I det seneste årti har har terapeutiske tilgange mod behandling af annonce fokuseret på forebyggelse af Aβ produktion3. Fleste undersøgelser har fokuseret på enten forøgelse af α-secretase aktivitet, som kan udelukke γ-secretase spaltning af APP og dermed mindske produktionen af Aβ eller hæmning af β-og/eller γ-secretase aktiviteter4. Desværre, ikke-selektiv hæmning af β - eller γ-secretases resultater i uundgåelige bivirkninger, der på grund af interferens med andre fysiologiske substrater af β - og γ-secretase5,6virkemåde. Med hensyn til γ-secretase hæmmere, har de seneste undersøgelser rapporteret opdagelsen af en række kemiske og genetiske modulatorer, der kan regulere Aβ produktion mens øve ubetydelig indvirkning på den væsentlige γ-secretase-medieret behandling af Notch7 ,8,9,10,11,12, men vellykket therapeutics er endnu ikke blevet udviklet. Således er yderligere systematisk skærme berettiget til at opdage nye genetiske og kemiske modifikatorer, der selektivt kan modulere γ-secretase-medieret APP behandling.
Γ-Secretase vides at kløve mere end 90 forskellige membran-forankrede proteiner. Blandt disse substrater er hak, hvis aktivitet er afgørende for celle skæbne beslutsomhed og differentiering under udvikling13. Selektivt modulerende γ-secretase-katalyseret APP forarbejdning i mangel af påvirker Notch behandling bliver afgørende for minimere Notch-relaterede bivirkninger af γ-secretase hæmmere, og denne selektivitet menes at være en primær faktor, der vil diktere den biologiske effekt af potentielle γ-secretase modulatorer til kronisk behandling af annonce. Der har været en række arylsulfonamide derivater, såsom GSI-953 (begacestat) og BMS-708163 (avagacestat), der er blevet fundet for at udstille potent og selektiv hæmning af γ-secretase14,15. En tidligere undersøgelse har vist, at den differentiale hæmning af γ-secretase spaltning af APP og hak kan observeres ved hjælp af en kvantitativ ELISA-baseret analyse i kombination med i vivo validering ved hjælp af zebrafisk16. Derudover er cellebaserede assay paradigmer etableret til at løse den potentielle substrat selektivitet af γ-secretase mod APP og hak17,18. Bruger protokoller ligner disse beskrevet heri, vi har for nylig opdaget en ny klasse af (D)-leucinamides, potent modulere γ-secretase spaltning af APP med Notch-besparende selektivitet19. Sammen, giver denne samling af undersøgelser en proof-of-concept støtte idéen om, at substrat selektivitet/tilgængelighed af γ-secretase kan være underlagt kemiske graduering og eksperimentel verifikation. Men, disse assay platforme ofte stole på relativt lav-overførselshastighed readouts og arbejdskrævende tilgange, der ikke opfylder muligvis industrielle standarder for drug discovery programmer.
Vi har for nylig genereret cellebaserede luciferase reporter gen assays, der kvantitativt kan afgøre den katalytiske aktivitet af γ-secretase mod to forskellige substrater, den 99-amino syre C-terminale fragment af APP (APP-C99) og den ekstracellulære domæne-slettet Notch peptid (NΔE). Både APP-C99 og NΔE har været meget anvendt som direkte substrater af γ-secretase i forskellige assays. For at generere homogen og sammenhængende luciferase reporter gen assays til γ-secretase kløvningen af enten APP-C99 eller NΔE, genereret vi en HEK-afledte stabil cellelinje (CG), som constitutively udtrykker en Gal4 promoter-drevet firefly luciferase reporter ( Gal4-Luc) og Gal4/VP16-tagget APP-C99 fusion protein (C99-GV). Derudover genereret vi en anden HEK-afledte stabil cellelinje (NG) som constitutively udtrykker Gal4-Luc og Gal4/VP16-mærket NΔE (NΔE-GV). Brug disse roman substrat-specifikke γ-secretase assays, give vi nu en metode til at kvantificere og skelne den katalytiske aktivitet af γ-secretase mod forskellige substrater (APP-C99 i CG celler), eller NΔE i NG celler. Derudover blev substrat-specifikke γ-secretase assays designet til at være befordrende for high-indhold screening platforme. Disse nyligt genererede γ-secretase assays kunne bane vejen for opdagelsen af roman γ-secretase modulatorer, som kunne forbedre kognitive funktion ved at undertrykke Aβ produktion uden at fremkalde uønskede Notch hæmning for næste generations behandling af annonce.

<table>
	<tbody>
		<tr>
			<td>VictorLight luminescence plate reader</td>
			<td>PerkinElmer</td>
			<td>2030-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Class II, Type 2 Biological Safety Cabinet</td>
			<td>Thermo Scientific</td>
			<td>1300 Series A2</td>
			<td></td>
		</tr>
		<tr>
			<td>CL-387,785</td>
			<td>EMD Calbiochem</td>
			<td>233100-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPT</td>
			<td>Merck</td>
			<td>565770</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher</td>
			<td>12100046</td>
			<td>main compnent of growth medium</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>Thermo Fisher</td>
			<td>A1113903</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeocin</td>
			<td>Thermo Fisher</td>
			<td>R25005</td>
			<td></td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>Gibco</td>
			<td>10687010</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytomer</td>
			<td>Sigma-Aldrich</td>
			<td>BR717805 Aldrich</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well microplate</td>
			<td>Nunc</td>
			<td>156545</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td>Sigma</td>
			<td>T7660</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Steady-Glo luciferase assay reagent</td>
			<td>Promega</td>
			<td>E2510</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidified CO2 incubator</td>
			<td>Revco</td>
			<td>Ultima II</td>
			<td></td>
		</tr>
		<tr>
			<td>T-REx293 cell line</td>
			<td>Invitrogen</td>
			<td>R71007</td>
			<td></td>
		</tr>
		<tr>
			<td>Wallac 1420 software version 3.0</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>instrument control software of the luminescence microplate reader</td>
		</tr>
	</tbody>
</table>

quantitative measurement of &#947;-secretase-mediated amyloid precursor protein and notch cleavage in cell-based luciferase reporter assay platforms

Vi har udviklet et par af celle-baseret reporter gen assays til kvantitativt måle γ-secretase spaltning af forskellige substrater. Dette manuskript beskriver procedurer, der kan bruges til at overvåge γ-secretase-medieret kløvningen af enten APP-C99 eller hak, ved hjælp af en Gal4 promoter-drevet firefly luciferase reporter system. Disse assays blev etableret af stabilt Co transfecting HEK293 celler med Gal4-drevet luciferase reporter gen og enten Gal4/VP16-mærket C-terminale fragmentet af APP (APP-C99; CG celler), eller Gal4/VP16-tagged Notch-ΔE (NΔE; NG celler). Ved hjælp af disse reporter assays parallelt, vi har vist, at en ErbB2-hæmmer, CL-387,785, fortrinsvis kan undertrykke γ-secretase spaltning af APP-C99 i CG celler, men ikke NΔE i NG celler. De differentierede svar udstillet af CG og NG celler, når de behandles med CL-387,785, repræsenterer en foretrukne karakteristisk for γ-secretase modulatorer, og disse svar er i skarp kontrast til pan-hæmning af γ-secretase induceret af DAPT. Vores undersøgelser giver direkte beviser at γ-secretase aktiviteter mod forskellige substrater kan differentieres i cellulære sammenhæng. Disse nye assays kan derfor være nyttige værktøjer i drug discovery for forbedret annonce terapier.

Hæmning af ErbB2 af CL-387,785 kan varierende fremme behandlingen af APP-C99 af Γ -secretase uden at påvirke Notch kavalergang For at etablere en celle-baserede analyse, som kvantitativt kan måle proteolytiske kløvningen af en bestemt γ-secretase substrat, genereret vi linjen HEK293-afledte CG celle af stabil Co Transfektion af en tetracyklin-inducerbar C-uhelbredeligt Gal4/VP16-mærket APP-C99 og en Gal4 promoter-drevet firefly ...

Watch this Scientific Journal Video about Quantitative Measurement of ÃŽÂ³-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms at JoVE.com

Quantitative Measurement of ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â³-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms

Kvantitativ måling af γ-Secretase-medieret Amyloid forløber Protein og hak kavalergang i celle-baserede Luciferase Reporter Assay platforme

Vi har med succes skabt to substrat-specifikke γ-secretase assays. Begge cellebaserede assays præsenteres her er designet til at kvantificere γ-secretase enzymaktiviteter via output af firefly luciferase journalister.

We have developed a pair of cell-based reporter gene assays to quantitatively measure &#947;-secretase cleavage of distinct substrates. This manuscript ...

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Neuroscience

Quantitative Measurement of &#947;-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms

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The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

High-throughput analyse af Mammale Olfaktoriske Receptors: Måling af receptor-aktivering via luciferaseaktivitet

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Påvisning af<em&gt; In Situ</em&gt; Protein-protein-komplekser på<em&gt; Drosophila</em&gt; Larve Neuromuskulær Junction Brug Proximity ligeringsassayet

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Stereotaktisk Infusion af Oligomert amyloid-beta i musen Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Kvantitativ måling af relative retinsyre Niveauer i E8.5 embryoner og neurosfære Cultures Brug af F9 RARE-LacZ Cell-baserede Reporter Assay

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Modeling Amyloid-ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Modellering Amyloid-β42 toksicitet og Neurodegeneration i voksen zebrafisk hjerne

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Assay udvikling for høje indhold kvantificering af Sod1 Mutant Protein samlede dannelse i levende celler

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Kvantitativ Autoradiografisk metode til bestemmelse af de regionale rater for cerebral protein syntese in vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it ...