Name: in vivo detection and analysis of rb protein sumoylation in human cells
Uploaded: 2017-11-02T13:00:00
Description: Watch this Scientific Journal Video about In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells at JoVE.com

Denne studien ble støttet av tilskudd fra vitenskap og teknologi Commission of Shanghai (Grant nr. 14411961800) og National Natural Science Foundation i Kina (Grant nr. 81300805).

1. gjenkjenning av endogene Rb SUMOylation på den tidlige G1 fasen <ol><li>celle kultur og celle syklus synkronisering.
	<ol><li>Opprettholde HEK293 celler i vekstmediet som inneholder Dulbecco &#39; s endret Eagle Medium (DMEM) med 1% penn-Strep og 10% fosterets bovin serum (FBS) på 37 ° C og 5% CO 2 i en inkubator.</li>
		<li>Synkronisere HEK293 cellene på G0 fasen.
		<ol><li>Antall HEK293 celler ved hjelp av en hemocytometer og seed ~1.5 x 10 7 celler i en 15 cm rett med 2...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retinoblastoma+protein+and+cell+cycle+control.">The retinoblastoma protein and cell cycle control.</a> Cell. 81 (3), 323-330 (1995).</li><li>Giacinti, C., Giordano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RB+and+cell+cycle+progression.">RB and cell cycle progression.</a> Oncogene. 25 (38), 5220-5227 (2006).</li><li>Burkhart, D. L., Sage, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+of+tumour+suppression+by+the+retinoblastoma+gene.">Cellular mechanisms of tumour suppression by the retinoblastoma gene.</a> Nat Rev Cancer. 8 (9), 671-682 (2008).</li><li>Ferreira, R., Naguibneva, I., Pritchard, L. L., Ait-Si-Ali, S., Harel-Bellan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rb/chromatin+connection+and+epigenetic+control:+opinion.">The Rb/chromatin connection and epigenetic control: opinion.</a> Oncogene. 20 (24), 3128-3133 (2001).</li><li>Hilgendorf, K. I., 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+retinoblastoma+protein+induces+apoptosis+directly+at+the+mitochondria.">The retinoblastoma protein induces apoptosis directly at the mitochondria.</a> Genes Dev. 27 (9), 1003-1015 (2013).</li><li>Munro, S., Carr, S. M., La Thangue, N. 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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=Hypo-phosphorylation+of+the+retinoblastoma+protein+(pRb)+by+cyclin+D:Cdk4/6+complexes+results+in+active+pRb.">Hypo-phosphorylation of the retinoblastoma protein (pRb) by cyclin D:Cdk4/6 complexes results in active pRb.</a> Proc Natl Acad Sci U S A. 94 (20), 10699-10704 (1997).</li><li>Ledl, A., Schmidt, D., Muller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+oncoproteins+E1A+and+E7+and+cellular+LxCxE+proteins+repress+SUMO+modification+of+the+retinoblastoma+tumor+suppressor.">Viral oncoproteins E1A and E7 and cellular LxCxE proteins repress SUMO modification of the retinoblastoma tumor suppressor.</a> Oncogene. 24 (23), 3810-3818 (2005).</li><li>Li, 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=Expression+of+SUMO-2/3+induced+senescence+through+p53-+and+pRB-mediated+pathways.">Expression of SUMO-2/3 induced senescence through p53- and pRB-mediated pathways.</a> J Biol Chem. 281 (47), 36221-36227 (2006).</li><li>Meng, F., Qian, J., Yue, H., Li, X., Xue, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMOylation+of+Rb+enhances+its+binding+with+CDK2+and+phosphorylation+at+early+G1+phase.">SUMOylation of Rb enhances its binding with CDK2 and phosphorylation at early G1 phase.</a> Cell Cycle. 15 (13), 1724-1732 (2016).</li><li>Geiss-Friedlander, R., Melchior, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concepts+in+sumoylation:+a+decade+on.">Concepts in sumoylation: a decade on.</a> Nat Rev Mol Cell Biol. 8 (12), 947-956 (2007).</li><li>Flotho, A., Melchior, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sumoylation:+a+regulatory+protein+modification+in+health+and+disease.">Sumoylation: a regulatory protein modification in health and disease.</a> Annu Rev Biochem. 82, 357-385 (2013).</li><li>Eifler, K., Vertegaal, A. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SENP1-modulated+sumoylation+regulates+retinoblastoma+protein+(RB)+and+Lamin+A/C+interaction+and+stabilization.">SENP1-modulated sumoylation regulates retinoblastoma protein (RB) and Lamin A/C interaction and stabilization.</a> Oncogene. 35 (50), 6429-6438 (2016).</li><li>Jakobs, 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=Ubc9+fusion-directed+SUMOylation+(UFDS):+a+method+to+analyze+function+of+protein+SUMOylation.">Ubc9 fusion-directed SUMOylation (UFDS): a method to analyze function of protein SUMOylation.</a> Nat Methods. 4 (3), 245-250 (2007).</li><li>Gareau, J. R., Lima, C. 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Dette dokumentet beskriver en protokoll for å finne og analysere endogene SUMOylation av Rb i menneskeceller. Som denne metoden er spesielt fokusert på endogene Rb protein uten noen veksling av globale SUMO-relaterte signal, er det et viktig verktøy for å undersøke Rb-SUMO modifisering under helt naturlig fysiologiske forhold.
For å oppnå dette, er det viktig å huske på at: 1) selv om SUMO omfatter fire isoformene (SUMO1-4, hver kodet av ulike gener) i forhold til ubiquitination, alle...

Nøyaktig kontroll av cellen syklus progresjon i eukaryote celler er basert på et stramt regulatoriske nettverk, som sikrer at spesielle hendelser skje i en organisert måte1,2. En av de viktigste aktørene i dette nettverket er retinoblastoma (Rb) protein, den første klonede tumor suppressor1,3. Rb protein er antatt å være en negativ regulator av cellen syklus progresjon, spesielt for G0/G1 S fase overgang og tumor vekst4,5. Svikt i Rb funksjon enten direkte fører til de vanligste intraokulært kreft hos barn, retinoblastoma, eller bidrar til utvikling av mange andre typer kreft5. Videre er Rb involvert i mange mobilnettet baner inkludert celledifferensiering og chromatin remodeling mitokondrier-mediert apoptose3,6,7.
Post-translasjonell modifikasjoner spille en sentral rolle i regulering av RB funksjon8,9. Fosforylering er en slik endring, og det fører vanligvis til Rb inaktivering. Quiescent G0 celler er Rb aktiv med lav fosforylering nivå. Som celler gjennom G0/G1 fase, er Rb sekvensielt hyper-fosforylert av en rekke cyclin-avhengige protein kinaser (CDKs) og cyclins, som cyclin E/CDK2 og cyclin D/CDK4/6, som deaktivere Rb og eliminere dens evne til å undertrykke celle-syklusen relaterte gene expression4,10. RB kan også endres av små ubiquitin-relaterte modifikator (SUMO)11,12,13.
SUMO er et ubiquitin som protein som covalently er knyttet til en rekke mål proteiner. Det er avgjørende for mangfoldig cellulære prosesser, inkludert celle syklus regulering, transkripsjon, protein mobilnettet lokalisering og fornedrelse, transport og DNA reparasjon14,15,16, 17 , 18. den SUMO Bøyning pathway består av dimeric SUMO E1 aktivering enzymet SAE1/UBA2, enkelt E2 conjugating enzymet Ubc9, flere E3 ligases og SUMO-spesifikke proteaser. Vanligvis må begynnende SUMO proteiner proteolytically behandles for å generere skjemaet modne. Eldre SUMO er aktivert av E1 heterodimer og deretter overført til E2 enzymet Ubc9. Endelig, C-terminalen glysin i SUMO covalently er konjugert til målet lysin av et substrat, og denne prosessen er vanligvis tilrettelagt av E3 ligases. SUMO protein kan fjernes fra modifisert underlaget ved bestemte proteaser. En tidligere studie av forfatterne av notatet avslørte at SUMOylation av Rb øker sin binding til CDK2, fører til hyper-fosforylering i den tidlige fasen G1, en prosess som er nødvendig for cellen syklus progresjon13. Vi viste også at tapet av Rb SUMOylation fører til en redusert celle spredning. Videre ble det nylig demonstrert at SUMOylation av Rb beskytter Rb protein fra proteasomal omsetning, dermed øker nivået av Rb protein i celler19. Derfor spiller SUMOylation en viktig rolle i Rb-funksjonen i ulike cellulære prosesser. For videre studier den funksjonelle konsekvens og fysiologiske relevans av Rb SUMOylation, er det viktig å utvikle en effektiv metode for å analysere SUMO status Rb i menneskelige celler og pasienten vev.
SUMOylation er en reversibel, svært dynamisk prosess. Dermed er det vanligvis vanskelig å oppdage SUMO endret proteiner under helt endogene forhold. Dette dokumentet presenterer en metode for å oppdage endogene Rb-SUMOylation. Videre, det viser hvordan oppdage eksogene Rb SUMOylation vill-type Rb og dens SUMO-mangelfull mutasjon11. Spesielt beskrevet Jacobs et al. en metode for å øke SUMO endring av en gitt substrat spesielt av Ubc9 fusion-rettet SUMOylation (UFDS)20. Denne protokollen basert på denne metoden, og beskriver hvordan å analysere Rb tvungen SUMOylation og funksjonelle konsekvensene. Gitt at hundrevis av SUMO underlag har blitt beskrevet tidligere og mer antatte SUMO underlag identifisert fra mange proteomic-baserte analyser, kan denne protokollen brukes til å analysere SUMO-endring av disse proteiner i humane celler.

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			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl70" style="width:89pt" width="119">11995065</td>
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			<td class="xl68" height="21" style="height:15.75pt">Opti-MEM&#160;</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl70" style="width:89pt" width="119">31985070</td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Fetal Bovine Serum (FBS)</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl70" style="width:89pt" width="119">26140079</td>
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			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Penicillin-Streptomycin</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl70" style="width:89pt" width="119">15140122</td>
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			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl70" style="width:89pt" width="119">10010023</td>
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			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Trypsin-EDTA</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl70" style="width:89pt" width="119">25200056</td>
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			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Thymidine</td>
			<td class="xl65" style="width:206pt" width="275">Sigma</td>
			<td class="xl70" style="width:89pt" width="119">T9250</td>
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			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Nocodazole</td>
			<td class="xl65" style="width:206pt" width="275">Sigma</td>
			<td class="xl70" style="width:89pt" width="119">M1404</td>
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			<td class="xl65" style="width:206pt" width="275">AMRESCO</td>
			<td class="xl70" style="width:89pt" width="119">694</td>
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			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">RNase A&#160;</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl70" style="width:89pt" width="119">EN0531</td>
			<td class="xl68"></td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">N-Ethylmaleimide</td>
			<td class="xl65" style="width:206pt" width="275">Sigma</td>
			<td class="xl70" style="width:89pt" width="119">E3876&#160;</td>
			<td class="xl68"></td>
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			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Sodium Dodecyl Sulfate (SDS)</td>
			<td class="xl65" style="width:206pt" width="275">AMRESCO</td>
			<td class="xl70" style="width:89pt" width="119">M107</td>
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			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Nonidet P-40 Substitute (NP-40)</td>
			<td class="xl65" style="width:206pt" width="275">AMRESCO</td>
			<td class="xl70" style="width:89pt" width="119">M158</td>
			<td class="xl68"></td>
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			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">protease inhibitor</td>
			<td class="xl65" style="width:206pt" width="275">Roche</td>
			<td class="xl71" style="width:89pt" width="119">5892970001</td>
			<td class="xl68"></td>
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			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Mouse Immunoglobulin G (IgG)</td>
			<td class="xl65" style="width:206pt" width="275">Santa Cruz Biotechnology</td>
			<td class="xl71" style="width:89pt" width="119">sc-2025</td>
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			<td class="xl68" height="21" style="height:15.75pt">Rb antibody</td>
			<td class="xl65" style="width:206pt" width="275">Cell Signaling Technology</td>
			<td class="xl71" style="width:89pt" width="119">#9309</td>
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			<td class="xl68" height="21" style="height:15.75pt">Protein A/G-Sepharose Beads</td>
			<td class="xl65" style="width:206pt" width="275">Santa Cruz Biotechnology</td>
			<td class="xl71" style="width:89pt" width="119">sc-2003</td>
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			<td class="xl68" height="21" style="height:15.75pt">Lipofectamine-2000&#160;</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl71" style="width:89pt" width="119">11668019</td>
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			<td class="xl65" height="23" style="width:275pt;height:17.25pt" width="366">Nickel Nitrilotriacetic Acid (Ni-NTA) Agarose Beads</td>
			<td class="xl65" style="width:206pt" width="275">Qiagen</td>
			<td class="xl71" style="width:89pt" width="119">30230</td>
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			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Imidazole</td>
			<td class="xl65" style="width:206pt" width="275">Sigma</td>
			<td class="xl71" style="width:89pt" width="119">I0250</td>
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			<td class="xl68" height="21" style="height:15.75pt">4%-20% Gradient SDS-PAGE Gel</td>
			<td class="xl65" style="width:206pt" width="275">BIO-RAD</td>
			<td class="xl71" style="width:89pt" width="119">4561096</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="height:15.75pt">Polyvinylidene Difluoride (PVDF) Membrane</td>
			<td class="xl65" style="width:206pt" width="275">Millipore</td>
			<td class="xl71" style="width:89pt" width="119">IPVH00010</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Tween-20</td>
			<td class="xl65" style="width:206pt" width="275">AMRESCO</td>
			<td class="xl71" style="width:89pt" width="119">M147</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Tubulin antibody</td>
			<td class="xl65" style="width:206pt" width="275">Abmart</td>
			<td class="xl71" style="width:89pt" width="119">M30109</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">SUMO1 antibody</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl71" style="width:89pt" width="119">33-2044</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">GFP antibody</td>
			<td class="xl65" style="width:206pt" width="275">Abmart</td>
			<td class="xl71" style="width:89pt" width="119">M20004</td>
			<td class="xl68"></td>
		</tr>
		<tr height="23" style="height:17.25pt">
			<td class="xl65" height="23" style="width:275pt;height:17.25pt" width="366">Horseradish Peroxidase (HRP) secondary antibody</td>
			<td class="xl65" style="width:206pt" width="275">Jackson ImmunoResearch Laboratories</td>
			<td class="xl71" style="width:89pt" width="119">715-035-150</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">enhanced chemiluminescence (ECL) Kit</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl71" style="width:89pt" width="119">32106</td>
			<td class="xl68"></td>
		</tr>
	</tbody>
</table>

in vivo detection and analysis of rb protein sumoylation in human cells

Post-translasjonell modifikasjoner av proteiner er avgjørende for riktig regulering av intracellulær signaltransduksjon. Blant endringene er små ubiquitin-relaterte modifikator (SUMO) en ubiquitin-lignende protein som tilhører covalently lysin rester av en rekke mål proteiner å regulere cellular prosesser, som gene transkripsjon, DNA-reparasjon, protein samhandling og fornedrelse, subcellular transport og signaltransduksjon. Den vanligste måten å oppdage protein SUMOylation er basert på uttrykk og rensing av rekombinant merket proteiner i bakterier, slik at en i vitro biokjemiske reaksjon som er enkel og egnet for adressering mekanistisk spørsmål. Men komplekse prosessen med SUMOylation i vivoer det mer utfordrende å finne og analysere protein SUMOylation i celler, spesielt når under endogene forhold. En fersk studie av forfatterne av notatet avdekket at endogene retinoblastoma (Rb) protein, en tumor suppressor som er viktig for negative regulering av cellen syklus progresjon, er spesielt SUMOylated på den tidlige G1-fasen. Dette dokumentet beskriver en protokoll for deteksjon og analyse av Rb SUMOylation under både endogene og ytre forhold i menneskeceller. Denne protokollen er egnet for phenotypical og funksjonelle etterforskningen av SUMO-endring av Rb, samt mange andre SUMO målrettede proteiner, menneskelige celler.

For å oppdage endogene Rb-SUMOylation under cellen syklus progresjon, synkronisert denne studien først HEK293 celler på fem ulike stadier av celle syklus (G0, tidlig G1, G1, S og G2/M) som beskrevet i delen protokollen i dette papiret. Kvaliteten på synkronisering ble bekreftet ved hjelp av nukleinsyre flekken propidium iodide etterfulgt av flyt cytometri analyse (figur 1). Deretter var cellene samlet og lysed av denaturing RIPA buffer. SUMO proteaser inh...

Watch this Scientific Journal Video about In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells at JoVE.com

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Små ubiquitin-relaterte modifikator (SUMO) familie proteiner er konjugert til lysin rester av målet proteiner til å regulere ulike cellulære prosesser. Dette dokumentet beskriver en protokoll for påvisning av retinoblastoma (Rb) protein SUMOylation under endogene og ytre forhold i menneskeceller.

I Vivo Deteksjon og analyse av Rb Protein SUMOylation i menneskeceller

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, ...

Research

JoVE Journal

Biology

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

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Effektiv Generation Menneskelig indusert pluripotent stamceller fra menneskelige somatiske celler med Sendai-virus

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 ...

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Profilering Individuell humane embryonale stamceller ved kvantitativ RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Protein Purification teknikk som gjør at Påvisning av Sumoylation og ubiquitinering av Spirende gjær kinetochore Proteiner Ndc10 og Ndc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

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Høy oppløsning Time-lapse Imaging og automatisk analyse av mikrotubulidynamikk i levende menneske navleveneendotel Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...