Name: co-immunoprecipitation assay using endogenous nuclear proteins from cells cultured under hypoxic conditions
Uploaded: 2018-08-02T14:30:00
Description: Watch this Scientific Journal Video about Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions at JoVE.com

Vi tackar Assoc. Prof. synd Tiong Ong för användning av hypoxi arbetsstationen. Detta arbete stöds av följande: Singapore undervisningsministeriet, MOE 1T1-02/04 och MOE2015-T2-2-087 (att Y.A.), Lee Kong Chian School of Medicine, Nanyang Technological University startpeng M4230003 (till P.O.B.), Vetenskapsrådet, den Familjen Erling-Perssons Stiftelse, Stiftelsen Novo Nordisk, Stichting af Jochnick Foundation, den svenska Diabetesförbundet, Scandia försäkringsbolaget, diabetesforskning och Wellnesss kaj von Kantzow stiftelse, den Strategiska forskningsprogrammet i Diabetes vid Karolinska Institutet, det Europeiska forskningsrådet ERC-2013-AdG 338936-Betalmage, och Knut och Alice Wallenbergs stiftelse.

Detta protokoll avsnitt, som använder mänskliga embryonala njure 293A (HEK293A) cell, s följer riktlinjerna i mänskliga forskningsetisk kommitté i Nanyang Technological University, Singapore.
1. induktion av hypoxi i HEK293A celler
<ol><li>Förbereda fyra 10 cm rätter och utsäde 3 – 5 x 106 HEK293A celler per maträtt i 10 mL Dulbecco's modified örnens medium (DMEM, 4,5 g/L glukos) kompletteras med 10% fetalt bovint serum (FBS), 2 mM L-glutamin, 110 mg/L natrium pyruvat,...

<ol>
	<li>Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factors+in+physiology+and+medicine.">Hypoxia-inducible factors in physiology and medicine.</a> Cell. 148 (3), 399-408 (2012).</li><li>Bartels, K., Grenz, A., Eltzschig, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+and+inflammation+are+two+sides+of+the+same+coin.">Hypoxia and inflammation are two sides of the same coin.</a> Proc Natl Acad Sci U S A. 110 (46), 18351-18352 (2013).</li><li>Jiang, B. H., Rue, E., Wang, G. L., Roe, R., Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimerization,+DNA+binding,+and+transactivation+properties+of+hypoxia-inducible+factor+1.">Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1.</a> J Biol Chem. 271 (30), 17771-17778 (1996).</li><li>Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF-1:+mediator+of+physiological+and+pathophysiological+responses+to+hypoxia.">HIF-1: mediator of physiological and pathophysiological responses to hypoxia.</a> J Appl Physiol. 88 (4), 1474-1480 (2000).</li><li>Kallio, P. 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=Signal+transduction+in+hypoxic+cells:+Inducible+nuclear+translocation+and+recruitment+of+the+CBP/p300+coactivator+by+the+hypoxia-inducible+factor-1alpha.">Signal transduction in hypoxic cells: Inducible nuclear translocation and recruitment of the CBP/p300 coactivator by the hypoxia-inducible factor-1alpha.</a> EMBO J. 17 (22), 6573-6586 (1998).</li><li>Ke, Q., Costa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factor-1+(HIF-1).">Hypoxia-inducible factor-1 (HIF-1).</a> Mol Pharmacol. 70 (5), 1469-1480 (2006).</li><li>Gustafsson, M. 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=Hypoxia+requires+notch+signaling+to+maintain+the+undifferentiated+cell+state.">Hypoxia requires notch signaling to maintain the undifferentiated cell state.</a> Dev Cell. 9 (5), 617-628 (2005).</li><li>Zheng, X., 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=Interaction+with+factor+inhibiting+HIF-1+defines+an+additional+mode+of+cross-coupling+between+the+Notch+and+hypoxia+signaling+pathways.">Interaction with factor inhibiting HIF-1 defines an additional mode of cross-coupling between the Notch and hypoxia signaling pathways.</a> Proc Natl Acad Sci U S A. 105 (9), 3368-3373 (2008).</li><li>D'Ignazio, L., Bandarra, D., Rocha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF-kappaB+and+HIF+crosstalk+in+immune+responses.">NF-kappaB and HIF crosstalk in immune responses.</a> FEBS J. 283 (3), 413-424 (2016).</li><li>Wang, G. L., Jiang, B. H., Rue, E. A., Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factor+1+is+a+basic-helix-loop-helix-PAS+heterodimer+regulated+by+cellular+O2+tension.">Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.</a> Proc Natl Acad Sci U S A. 92 (12), 5510-5514 (1995).</li><li>Zheng, X., 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=Cell-type-specific+regulation+of+degradation+of+hypoxia-inducible+factor+1+alpha:+Role+of+subcellular+compartmentalization.">Cell-type-specific regulation of degradation of hypoxia-inducible factor 1 alpha: Role of subcellular compartmentalization.</a> Mol Cell Biol. 26 (12), 4628-4641 (2006).</li><li>Depping, 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=Nuclear+translocation+of+hypoxia-inducible+factors+(HIFs):+involvement+of+the+classical+importin+alpha/beta+pathway.">Nuclear translocation of hypoxia-inducible factors (HIFs): involvement of the classical importin alpha/beta pathway.</a> Biochim Biophys Acta. 1783 (3), 394-404 (2008).</li><li>Wei, H., 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=Hypoxia+induces+oncogene+yes-associated+protein+1+nuclear+translocation+to+promote+pancreatic+ductal+adenocarcinoma+invasion+via+epithelial-mesenchymal+transition.">Hypoxia induces oncogene yes-associated protein 1 nuclear translocation to promote pancreatic ductal adenocarcinoma invasion via epithelial-mesenchymal transition.</a> Tumour Biol. 39 (5), (2017).</li><li>Chang, H. 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=Hypoxia+promotes+nuclear+translocation+and+transcriptional+function+in+the+oncogenic+tyrosine+kinase+RON.">Hypoxia promotes nuclear translocation and transcriptional function in the oncogenic tyrosine kinase RON.</a> Cancer Res. 74 (16), 4549-4562 (2014).</li><li>Moriya, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+nature+of+overexpression+experiments.">Quantitative nature of overexpression experiments.</a> Mol Biol Cell. 26 (22), 3932-3939 (2015).</li><li>Prelich, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+overexpression:+Uses,+mechanisms,+and+interpretation.">Gene overexpression: Uses, mechanisms, and interpretation.</a> Genetics. 190 (3), 841-854 (2012).</li><li>Zheng, X., 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=A+Notch-independent+mechanism+contributes+to+the+induction+of+Hes1+gene+expression+in+response+to+hypoxia+in+P19+cells.">A Notch-independent mechanism contributes to the induction of Hes1 gene expression in response to hypoxia in P19 cells.</a> Exp Cell Res. 358 (2), 129-139 (2017).</li><li>Farris, M. H., Ford, K. A., Doyle, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualitative+and+quantitative+assays+for+detection+and+characterization+of+protein+antimicrobials.">Qualitative and quantitative assays for detection and characterization of protein antimicrobials.</a> J Vis Exp. (110), e53819 (2016).</li><li>Chilov, 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=Induction+and+nuclear+translocation+of+hypoxia-inducible+factor-1+(HIF-1):+heterodimerization+with+ARNT+is+not+necessary+for+nuclear+accumulation+of+HIF-1alpha.">Induction and nuclear translocation of hypoxia-inducible factor-1 (HIF-1): heterodimerization with ARNT is not necessary for nuclear accumulation of HIF-1alpha.</a> J Cell Sci. 112 (Pt 8), 1203-1212 (1999).</li><li>Yin, 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=Arylsulfonamide+KCN1+inhibits+in+vivo+glioma+growth+and+interferes+with+HIF+signaling+by+disrupting+HIF-1alpha+interaction+with+cofactors+p300/CBP.">Arylsulfonamide KCN1 inhibits in vivo glioma growth and interferes with HIF signaling by disrupting HIF-1alpha interaction with cofactors p300/CBP.</a> Clin Cancer Res. 18 (24), 6623-6633 (2012).</li><li>Holmquist-Mengelbier, 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=Recruitment+of+HIF-1alpha+and+HIF-2alpha+to+common+target+genes+is+differentially+regulated+in+neuroblastoma:+HIF-2alpha+promotes+an+aggressive+phenotype.">Recruitment of HIF-1alpha and HIF-2alpha to common target genes is differentially regulated in neuroblastoma: HIF-2alpha promotes an aggressive phenotype.</a> Cancer Cell. 10 (5), 413-423 (2006).</li><li>Koh, M. 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HIF-1 komplexet är en mästare regulator av cellulära syre homeostas och reglerar en uppsjö av gener involverade i olika cellulära adaptiva svar till hypoxi. Identifiering av romanen HIF-1 samverkande proteiner är viktiga för förståelsen av hypoxisk signaltransduktion. Co-immunoprecipitation experiment används ofta för protonpumpshämmare studier för att avgränsa cellulära cellsmedierade reaktioner. Men protein överuttryck används fortfarande flitigt och detta kan leda till experimentella artefakter. Dessu...

Hypoxi uppstår när otillräcklig syre levereras till celler och vävnader i kroppen. Det spelar en avgörande roll i olika fysiologiska och patologiska processer såsom stamcellers differentiering, inflammation och cancer1,2. Hypoxi-inducerbara faktorer (HIFs) fungera som heterodimerer består av en syre-reglerade α-subenheten och ett konstitutivt uttryckta β subenhet kallas även ARNT3. Tre isoformer av HIF-α subunitsna (HIF-1α, HIF-2α och HIF-3α) och tre HIF-β-subenheter (ARNT/HIF-1β, ARNT2 och ARNT3) har identifierats hittills. HIF-1α och ARNT uttrycks ubiquitously, medan HIF-2α, HIF-3α, ARNT2 och ARNT3 har mer begränsad uttryck mönster4. HIF-1 protein komplex är den viktigaste regulatorn av hypoxi svaret. Hypoxisk villkor, HIF-1α blir stabiliserad, sedan translocates till kärnan och dimerizes med ARNT5. Därefter, detta komplex binder till specifika nukleotider kallas hypoxi responsive element (HREs) och reglerar uttrycket av målgener som är inblandade i olika processer inklusive angiogenes, erytropoesen och glykolys6. Utöver detta ”kanoniska” svar, den hypoxi signalvägen är också känt att överhörning med flera cellulära svar signalering vägar såsom Notch och Nuclear Factor-kappa B (NF-κB)7,8,9.
Identifiering av Roman HIF-1 samverkande proteiner är viktigt för en bättre förståelse för den hypoxi signalering utbildningsavsnitt. I motsats till ARNT, som är okänslig för syrehalten och konstitutivt uttryckta, är HIF-1α proteinnivåer hårt reglerad av cellulära syrenivåer. På normoxia (21% syre) är HIF-1α proteiner snabbt försämrade10,11. Den korta halveringstiden för HIF-1α på normoxia presenterar specifika tekniska utmaningar för detektion av proteinet från cell extrakt, liksom för identifiering av HIF-1α-interagera proteiner. Dessutom translocate flera transkriptionsfaktorer inklusive komplexet HIF-1 in i cellkärnan enligt hypoxisk villkor12,13,14. De flesta av de nuvarande metoderna för PPI studier utförs med icke-fysiologiskt överuttryck av proteiner. Sådan protein överuttryck har rapporterats orsaka olika cellulära defekter genom flera mekanismer inklusive resurs överbelastning, stökiometriska obalans, promiskuösa interaktioner och signalvägen moduleringen15,16. När det gäller PPI studier, kan protein överuttryck leda till falskt positiva, eller ens falska negativa, resultat beroende på protein egenskaper och funktioner av överuttryckt proteiner. Därför har de nuvarande metoderna för PPI studier ska ändras för att avslöja de fysiologiskt relevanta protonpumpshämmarna hypoxisk villkor. Vi har tidigare visat samspelet mellan HIF-1 och den Ets familj transkriptionsfaktor GA-bindande protein (GABP) i hypoxisk P19 celler, vilket bidrar till svaret från Hes1 arrangören till hypoxi17. Här beskriver vi ett co-immunoprecipitation protokoll för att studera protonpumpshämmare mellan endogena nukleära proteiner hypoxisk villkor. Samspelet mellan HIF-1α och ARNT visas som ett proof of concept. Detta protokoll är lämplig för att påvisa interaktioner mellan transkriptionsfaktorer och transkriptionell samtidig tillsynsmyndigheter hypoxisk villkor, inklusive men inte begränsat till identifiering av HIF-1 samverkande proteiner.

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			<td height="21" style="height:21px;width:279px;">Material</td>
			<td style="width:161px;"></td>
			<td style="width:135px;"></td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">1.0 M Tris-HCl Buffer, pH 7.4&#160;</td>
			<td style="width:161px;">1st BASE</td>
			<td style="width:135px;">1415</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Protein A/G Sepharose beads</td>
			<td style="width:161px;">Abcam</td>
			<td style="width:135px;">ab193262</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Natural Mouse IgG protein</td>
			<td style="width:161px;">Abcam</td>
			<td style="width:135px;">ab198772</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">EDTA</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1610729</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">2x Laemmli Sample Buffer</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1610737</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">2-Mercaptoethanol</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1610710</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Nitrocellulose Membrane &#160;&#160;</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1620112</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Blotting-Grade Blocker</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1706404</td>
			<td style="width:229px;">Non-fat dry milk for western blotting applications</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">10x Tris Buffered Saline (TBS)</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1706435</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">10% Tween 20</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1610781</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">10x Tris/Glycine/SDS</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1610732</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">10x Tris/Glycine Buffer&#160;</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1610771</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Precision Plus Protein Dual Color Standards</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1610374</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Anti-rabbit IgG, HRP-linked Antibody</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:135px;">7074</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Anti-mouse IgG, HRP-linked Antibody&#160;</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:135px;">7076</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">SignalFire ECL Reagent</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:135px;">6883</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td style="width:161px;">Corning</td>
			<td style="width:135px;">21-030-CV</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Phenylmethylsulfonyl fluoride (PMSF)</td>
			<td style="width:161px;">Merck Millipore</td>
			<td style="width:135px;">52332</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">ARNT/HIF-1 beta Antibody&#160;</td>
			<td style="width:161px;">Novus Biologicals</td>
			<td style="width:135px;">NB100-124&#160;</td>
			<td style="width:229px;">Concentration: 1.4 mg/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">HIF-1 alpha Antibody</td>
			<td style="width:161px;">Novus Biologicals</td>
			<td style="width:135px;">NB100-479</td>
			<td style="width:229px;">Concentration: 1.0 mg/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">YY1 Antibody</td>
			<td style="width:161px;">Novus Biologicals</td>
			<td style="width:135px;">NBP1-46218</td>
			<td style="width:229px;">Concentration: 0.2 mg/mL</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Qproteome Nuclear Protein Kit</td>
			<td style="width:161px;">Qiagen</td>
			<td style="width:135px;">37582</td>
			<td style="width:229px;">Lysis buffer NL and Extraction Buffer NX1 are provied in the kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">GAPDH Antibody</td>
			<td style="width:161px;">Santa Cruz</td>
			<td style="width:135px;">sc-47724</td>
			<td style="width:229px;">Concentration: 0.2 mg/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Glycerol (&#8805;99%)</td>
			<td style="width:161px;">Sigma</td>
			<td style="width:135px;">G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Potassium chloride</td>
			<td style="width:161px;">Sigma</td>
			<td style="width:135px;">P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">RIPA buffer</td>
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</table>

co-immunoprecipitation assay using endogenous nuclear proteins from cells cultured under hypoxic conditions

Låga syrenivåer (hypoxi) utlöser en mängd adaptiv svaren med hypoxi-inducerbara faktorn 1 (HIF-1) komplex egenskap en mästare regulator. HIF-1 består av en heterodimeriskt syre-reglerade α-subenheten (HIF-1α) och konstitutivt uttryckta β subenhet (HIF-1β) kallas även aryl kolväte receptor nukleära translocator (ARNT), reglerar genernas olika processer inklusive angiogenes , erytropoesen och glykolys. Identifiering av HIF-1 samverkande proteiner är nyckeln till förståelsen av den hypoxi signalering utbildningsavsnitt. Förutom regleringen av HIF-1α stabilitet utlöser hypoxi också på nukleära flyttning av många transkriptionsfaktorer inklusive HIF-1α och ARNT. Särskilt, är de flesta av de nuvarande metoder att studera sådana protein-protein interaktioner (PPI) baserade på system där proteinnivåer är artificiellt ökade genom protein överuttryck. Protein överuttryck ofta leder till icke-fysiologiskt resultat uppkommer tidsmässiga och rumsliga artefakter. Här beskriver vi en modifierad co-immunoprecipitation protokoll efter hypoxi behandling med hjälp av endogena nukleära proteiner, och som ett proof of concept, att Visa samspelet mellan HIF-1α och ARNT. I detta protokoll, hypoxisk celler skördats hypoxisk villkor och den Dulbecco's Phosphate-Buffered Saline (DPBS) tvättbuffert var också pre skakad hypoxisk förhållanden före användning för att minska proteinnedbrytning eller proteinkomplex dissociation under reoxygenation. Dessutom extraherades de nukleära fraktionerna därefter för att koncentrera sig och stabilisera endogena nukleära proteiner och undvika möjliga falska resultat ses ofta under protein överuttryck. Detta protokoll kan användas för att demonstrera endogena och infödda interaktioner mellan transkriptionsfaktorer och transkriptionell samtidig tillsynsmyndigheter hypoxisk villkor.

För att bedöma den cellulära svaren på hypoxi, uttrycket nivåer och subcellulär lokalisering av komponenterna i HIF-1 komplexa följande hypoxi behandling undersöktes. HEK293A celler var odlade hypoxisk villkor för 4 h eller hålls på normoxia som kontroller. HIF-1α och ARNT proteinnivåer undersöktes i hela cellen eller kärn/cytoplasma extrakt genom western blot. Som förväntat, totala HIF-1α nivåer var uppreglerad av hypoxi, medan ARNT i totala cellular lysates inte var s...

Watch this Scientific Journal Video about Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions at JoVE.com

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

Här beskriver vi ett co-immunoprecipitation protokoll för att studera protein-protein interaktioner mellan endogena nukleära proteiner hypoxisk villkor. Denna metod är lämplig för demonstration av samspelet mellan transkriptionsfaktorer och transkriptionell samtidig regulatorer vid hypoxi.

Co-immunoprecipitation Assay med endogena nukleära proteiner från celler odlade hypoxisk villkor

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. ...

Research

JoVE Journal

Biology

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