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. ...

This method can help answer key questions in the hypoxic field, such as endogenous and native interactions between transcription factors and their transcriptional coregulators under hypoxic conditions. The main advantage of this technique is that the hypoxic cells are harvested under hypoxic conditions and the nuclear fractions are used for the co-immunoprecipitation. This protocol uses human embryonic kidney 293A cells grown in four 10-centimeter dishes.Seed three to five times 10 to the sixth cells per dish in 10 milliliters of Dulbeco's modified Eagle's medium supplemented with 10%FBS, L-glutamine, sodium pyruvate, penicillin and streptomycin. Culture the cells in a 37 degrees Celsius 5%carbon dioxide incubator. Twenty-four hours after seeding, when the cells have reached 80 to 90%confluency, put two dishes into the hypoxia subchamber in the incubator glovebox with 1%oxygen and 5%carbon dioxide at 37 degrees Celsius for four hours.Keep the other two dishes at normoxia. On the day before harvesting the hypoxia-treated cells, pre-equilibrate DPBS to hypoxic conditions by placing an uncovered 100 milliliter experiment glass reagent bottle filled with DPBS in the hypoxia subchamber for 24 hours. Approximately one hour prior to harvesting the cells, place an icebox containing ice into the processing chamber of the glovebox, which has been equilibrated to 1%oxygen and 5%carbon dioxide.Transfer the bottle containing the pre-equilibrated hypoxic DPBS from the hypoxia subchamber to the processing chamber and place it on ice. Four hours following hypoxia treatment, transfer the cells from the hypoxia subchamber to the processing chamber that has been pre-equilibrated to 1%oxygen and 5%carbon dioxide. Remove the culture medium by aspiration and with a 10 milliliter pipette, rinse the cells once with 10 milliliters of ice-cold pre-equilibrated hypoxic DPBS.Using a five milliliter pipette, add 5 milliliters of ice-cold pre-equilibrated DPBS and dislodge the cells by scraping with a cell scraper. Tilt the cell culture plate, collect the detached cells using a 10 milliliter pipette and transfer the cell suspension into a 15 milliliter conical tube. Keep the tube of cells on ice.Open the door between the processing and buffer chambers in the glovebox, both of which have been pre-equilibrated to 1%oxygen and 5%carbon dioxide. Transfer the 15 milliliter conical tubes on ice containing hypoxia-treated cells from the processing chamber to the buffer chamber. Open the door of the buffer chamber and remove the cells completely from the glovebox.Pellet both the hypoxic cells and the normoxic cells by centrifugation at 1000 times g for five minutes at four degrees Celsius. A nuclear extraction kit will be used for this procedure. Start by gently resuspending each cell pellet in 500 microliters of lysis buffer NL, supplemented with 1X protease inhibitor cocktail in 0.1 molar DTT by pipetting up and down several times.Add 25 microliters of detergent solution NP to the cell suspension and vortex for 10 seconds at maximum speed. Centrifuge at 1000 times 10 g at four degrees Celsius for five minutes. Save both the supernatant and the nuclear pellet.After collecting and storing the supernatant as described in the text protocol, resuspend the pellet-containing cell nucleii in 500 microliters of lysis buffer NL, supplemented with 1X protease inhibitor cocktail and 0.1 molar DTT by vortexing for five seconds at maximum speed. Resuspend the nuclear pellet in 50 microliters of extraction buffer NX1, supplemented with 1X protease inhibitor cocktail by pipetting the pellet up and down several times. Incubate on ice for 30 minutes.Every five minutes, vortex for 10 seconds at maximum speed. Centrifuge at 12, 000 x g at four degrees Celsius for 10 minutes. Collect the supernatant and transfer into mini dialysis devices with the maximum volume of 100 microliters per unit for desalting.Cap the mini dialysis devices and place them in a flotation device. Put the flotation device in a beaker containing 500 milliliters of pre-chilled dialysis buffer. Be careful to avoid direct contact with skin or inhalation of the dialysis buffer, because it contains PMSF.Incubate at four degrees Celsius with gentle stirring for 30 minutes. After 30 minutes, collect the samples from the corner of the mini dialysis devices and transfer to new 1.5 milliliter microcentrifuge tubes. Centrifuge at 12 thousand times g at four degrees Celsius for 10 minutes.Aliquot 25 microliters of each supernatant into a 1.5 microliter centrifuge tube and store at minus 80 degrees Celsius. Prepare the protein AG sepharose beads to be used in the immunopreciptation. Pipette 50 microlitera of beads into each of four 1.5 milliliter microcentrifuge tubes and add 500 microliters of DPBS buffer per tube.Pellet the beads by centrifugation. Discard the supernatant and resuspend the beads in 100 microliters of TBS buffer. Add two microliters of mouse monoclonal anti-ARNT antibody or mouse immunoglobulin G that was prepared by reconstituting 0.7 milligrams of mouse IgG in 500 microliters of TBS buffer.Take the microcentrifuge tubes containing the beads into a cold room. Place them in a tube rotator and incubate at 10 rpm for two hours. After two hours, pellet the beads by centrifugation and discard the supernatants.Dilute 200 micrograms of the nuclear protein lysate prepared earlier in 800 microliters of immunoprecipitation buffer. Incubate the lysate with the antibody-coupled beads at four degrees Celsius overnight. On the following day, pellet the beads by centrifugation at 3000 times g for two minutes at four degrees Celsius.Discard the supernatant. Add one milliliter of ice-cold TBS containing 0.2%NP-40 to each tube and spin down. In this manner, wash the beads three times with ice-cold TBS containing 0.2%NP-40.Boil the beads in 50 microliters of Laemmli sample buffer at 95 degrees Celsius for five minutes. Centrifuge the beads at 10 thousand times g at four degrees Celsius for five minutes. Collect the supernatant and discard the beads.The expression levels in subcellular localization of the components of the HIF-1 complex following hypoxia treatment were examined in HEK293A cells. As expected, total HIF-1a levels were upregulated by hypoxia whereas ARNT levels in total cellular lysates were not significantly altered. In addition, hypoxia induced nuclear accumulation of both HIF-1a and ARNT.Co-immunoprecipitation experiments were performed using nuclear extracts from HEK293A cells exposed to normoxic or hypoxic conditions. As shown here, HIF-1a was co-immunoprecipitated together with ARNT from the nuclear extracts of hypoxic HEK293A cells, indicating that HIF-1a interacts with ARNT under hypoxic conditions. After its development, this technique paved the way for researchers in the field of hypoxia to explore endogenous nuclear protein protein interactions and their hypoxic conditions.

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