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

We thank Assoc. Prof. Sin Tiong Ong for the use of the hypoxia workstation. This work was supported by the following: Singapore Ministry of Education, MOE 1T1-02/04 and MOE2015-T2-2-087 (to Y.A.), Lee Kong Chian School of Medicine, Nanyang Technological University start-up grant M4230003 (to P.O.B.), the Swedish Research Council, the Family Erling-Persson Foundation, the Novo Nordisk Foundation, the Stichting af Jochnick Foundation, the Swedish Diabetes Association, the Scandia Insurance Company, the Diabetes Research and Wellness Foundation, Berth von Kantzow's Foundation, the Strategic Research Program in Diabetes at Karolinska Institutet, the ERC ERC-2013-AdG 338936-Betalmage, and the Knut and Alice Wallenberg Foundation.

This protocol section, which uses human embryonic kidney 293A (HEK293A) cell,s follows the guidelines of human research ethics committee in Nanyang Technological University, Singapore.
1. Induction of Hypoxia in HEK293A Cells
<ol>
	<li>Prepare four 10 cm dishes and seed 3&#8211;5 x 106 HEK293A cells per dish in 10 mL Dulbecco&#39;s modified Eagle&#39;s medium (DMEM, 4.5 g/L glucose) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 110 mg/L sodium pyruvate, 100 U/m...

<ol>
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The HIF-1 complex is a master regulator of cellular oxygen homeostasis and regulates a plethora of genes involved in different cellular adaptive responses to hypoxia. Identification of novel HIF-1 interacting proteins is important for the understanding of hypoxic signal transduction. Co-immunoprecipitation experiments are commonly used for PPIs studies to delineate cellular signal transduction pathways. However, protein overexpression is still widely used and this may lead to experimental artifacts. In addition, HIF-1&#9...

Hypoxia occurs when inadequate oxygen is supplied to the cells and tissues of the body. It plays a critical role in various physiological and pathological processes such as stem cell differentiation, inflammation and cancer1,2. Hypoxia-inducible factors (HIFs) function as heterodimers composed of an oxygen-regulated &#945; subunit and a constitutively expressed &#946; subunit also known as ARNT3. Three isoforms of the HIF-&#945; subunits (HIF-1&#945;, HIF-2&#945; and HIF-3&#945;) and three HIF-&#946; subunits (ARNT/HIF-1&#946;, ARNT2 and ARNT3) have been identified to date. HIF-1&#945; and ARNT are ubiquitously expressed, whereas HIF-2&#945;, HIF-3&#945;, ARNT2 and ARNT3 have more restricted expression patterns4. The HIF-1 protein complex is the key regulator of the hypoxia response. Under hypoxic conditions, HIF-1&#945; becomes stabilized, then translocates to the nucleus and dimerizes with ARNT5. Subsequently, this complex binds to specific nucleotides known as hypoxia responsive elements (HREs) and regulates the expression of target genes involved in diverse processes including angiogenesis, erythropoiesis and glycolysis6. In addition to this &#34;canonical&#34; response, the hypoxia signaling pathway is also known to crosstalk with multiple cellular response signaling pathways such as Notch and Nuclear Factor-kappa B (NF-&#954;B)7,8,9.
The identification of novel HIF-1 interacting proteins is important for a better understanding of the hypoxia signaling pathway. In contrast to ARNT, which is insensitive to oxygen levels and constitutively expressed, HIF-1&#945; protein levels are tightly regulated by cellular oxygen levels. At normoxia (21% oxygen), HIF-1&#945; proteins are rapidly degraded10,11. The short half-life of HIF-1&#945; at normoxia presents specific technical challenges for the detection of the protein from cell extracts, as well as for the identification of HIF-1&#945;-interacting proteins. Furthermore, several transcription factors including those of the HIF-1 complex translocate into the nucleus under hypoxic conditions12,13,14. Most of the current methods used for PPI studies are performed using non-physiological overexpression of proteins. Such protein overexpression has been reported to cause different cellular defects through multiple mechanisms including resource overload, stoichiometric imbalance, promiscuous interactions, and pathway modulation15,16. In terms of PPI studies, protein overexpression can lead to false positive, or even false negative, results depending on the protein properties and functions of the overexpressed proteins. Therefore, the current methods for PPI studies have to be modified in order to reveal the physiologically relevant PPIs under hypoxic conditions. We have previously demonstrated the interaction between HIF-1 and the Ets family transcription factor GA-binding protein (GABP) in hypoxic P19 cells, which contributes to the response of the Hes1 promoter to hypoxia17. Here, we describe a co-immunoprecipitation protocol to study PPIs between endogenous nuclear proteins under hypoxic conditions. The interaction between HIF-1&#945; and ARNT is shown as a proof of concept. This protocol is suitable for demonstrating the interactions between transcription factors and transcriptional co-regulators under hypoxic conditions, including but not limited to the identification of HIF-1 interacting proteins.

<table border="0" cellpadding="0" cellspacing="0" width="803">
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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>
			<td style="width:161px;">Sigma</td>
			<td style="width:135px;">R0278</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Sodium Chloride (NaCl)</td>
			<td style="width:161px;">Sigma</td>
			<td style="width:135px;">71376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">NP-40</td>
			<td style="width:161px;">Sigma</td>
			<td style="width:135px;">127087-87-0</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM, 4.5 g/L glucose)</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">11995065</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Dithiothreitol (DTT)</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">R0861</td>
			<td></td>
		</tr>
		<tr height="42">
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co-immunoprecipitation assay using endogenous nuclear proteins from cells cultured under hypoxic conditions

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. HIF-1 consists of a heterodimeric oxygen-regulated &#945; subunit (HIF-1&#945;) and constitutively expressed &#946; subunit (HIF-1&#946;) also known as aryl hydrocarbon receptor nuclear translocator (ARNT), regulating genes involved in diverse processes including angiogenesis, erythropoiesis and glycolysis. The identification of HIF-1 interacting proteins is key to the understanding of the hypoxia signaling pathway. Besides the regulation of HIF-1&#945; stability, hypoxia also triggers the nuclear translocation of many transcription factors including HIF-1&#945; and ARNT. Notably, most of the current methods used to study such protein-protein interactions (PPIs) are based on systems where protein levels are artificially increased through protein overexpression. Protein overexpression often leads to non-physiological results arising from temporal and spatial artifacts. Here we describe a modified co-immunoprecipitation protocol following hypoxia treatment using endogenous nuclear proteins, and as a proof of concept, to show the interaction between HIF-1&#945; and ARNT. In this protocol, the hypoxic cells were harvested under hypoxic conditions and the Dulbecco's Phosphate-Buffered Saline (DPBS) wash buffer was also pre-equilibrated to hypoxic conditions before usage to mitigate protein degradation or protein complex dissociation during reoxygenation. In addition, the nuclear fractions were subsequently extracted to concentrate and stabilize endogenous nuclear proteins and avoid possible spurious results often seen during protein overexpression. This protocol can be used to demonstrate endogenous and native interactions between transcription factors and transcriptional co-regulators under hypoxic conditions.

To assess the cellular response to hypoxia, the expression levels and subcellular localization of the components of the HIF-1 complex following hypoxia treatment were examined. HEK293A cells were cultured under hypoxic conditions for 4 h or kept at normoxia as controls. HIF-1&#945; and ARNT protein levels were examined in whole cell or nuclear/cytoplasmic extracts by western blot. As expected, total HIF-1&#945; levels were upregulated by hypoxia, whereas ARNT levels in total cellular lysa...

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

Here we describe a co-immunoprecipitation protocol to study protein-protein interactions between endogenous nuclear proteins under hypoxic conditions. This method is suitable for demonstration of the interactions between transcription factors and transcriptional co-regulators at hypoxia.

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

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