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/mL penicillin and 100 mg/mL streptomycin. Culture the cells in a 37 &#176;C, 5% CO2 incubator. 
	NOTE: Cell seeding should be carried out in a biological safety cabinet (BSC). The surfaces of all materials to be placed in the BSC should be wiped with 70% ethanol.</li>
	<li>24 h after seeding, when the cells have reached 80&#8211;90% confluency, put two dishes into the hypoxia subchamber in the incubator glovebox (see Table of Materials) with 1% O2 and 5% CO2 at 37 &#176;C, and keep the other two dishes at normoxia (21% O2, 5% CO2 at 37 &#176;C) for 4 h. Use one set of normoxic and hypoxic cells to evaluate the hypoxia treatment by western blot and utilize the other set of cells for the co-immunoprecipitation experiments. 
	NOTE: The oxygen levels can be set at 0&#8211;5%, and the duration of the hypoxia treatment can be varied depending on the cell type and study objective.</li>
</ol>
2. Whole Cell and Nuclear Extraction
NOTE: See Table 1 for information on buffers used in this protocol.
<ol>
	<li>Harvest the normoxia control cells.
	<ol>
		<li>Remove the culture media by aspiration and rinse the cells with 10 mL DPBS (PH 7.0&#8211;7.2) using a 10 mL pipette. 
		NOTE: Avoid touching the cell monolayer with the pipette. During the washing, gently pipette the DPBS down the wall of the cell culture plate to avoid cell loss.</li>
		<li>Pipette 5 mL ice-cold DPBS into the plate and scrape the cells off the surface of the plate in ice-cold PBS with a cell scraper.</li>
		<li>Transfer the cell suspension into 15 mL conical tubes and keep on ice.</li>
	</ol>
	</li>
	<li>Harvest the cells cultured under hypoxic conditions.
	<ol>
		<li>Pre-equilibrate the DPBS to hypoxic conditions by placing an uncovered 100 mL experiment glass reagent bottle filled with DPBS in the hypoxia subchamber (1% O2 and 5% CO2 at 37 &#176;C) for 24 h in advance.</li>
		<li>Approximately 1 h prior to harvesting the hypoxia treated cells, place an ice box containing ice into the processing chamber of the glovebox, which has been equilibrated to 1% O2 and 5% CO2. Transfer the bottle containing the pre-equilibrated hypoxic DPBS from the hypoxia subchamber to the processing chamber and place it on ice.</li>
		<li>4 h following hypoxia treatment, transfer the cells from the hypoxia subchamber to the processing chamber that has been pre-equilibrated to 1% O2 and 5% CO2.</li>
		<li>Remove the culture media by aspiration and rinse the cells once with 10 mL ice-cold pre-equilibrated hypoxic DPBS with a 10 mL pipette.</li>
		<li>Add 5 mL ice-cold pre-equilibrated DPBS with a 5 mL pipette and dislodge the cells by scraping with a cell scraper.</li>
		<li>Tilt the cell culture plate and collect the detached cells using a 10 mL pipette. Transfer the cell suspension in DPBS into 15 mL conical tubes and keep on ice.</li>
		<li>Open the door between the processing and buffer chambers of the glovebox, both of which have been pre-equilibrated to 1% O2 and 5% CO2. Transfer the 15 mL 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.</li>
	</ol>
	</li>
	<li>Pellet both the normoxic cells from 2.1 and the hypoxic cells from 2.2 by centrifugation at 1,000 x g for 5 min at 4 &#176;C.</li>
	<li>Prepare the whole cell extracts.
	<ol>
		<li>Resuspend the cell pellets in 500 &#181;L of ice-cold radio immunoprecipitation assay (RIPA) lysis buffer containing 50 mM Trisaminomethane Hydrochloride (Tris-HCl) pH 8.0, 150 mM sodium chloride (NaCl), 1% tergitol-type NP-40 (NP-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS), supplemented with 1x protease inhibitor cocktail (see Table of Materials) by pipetting the cell pellets up and down several times.</li>
		<li>Transfer the cell lysates into new 1.5 mL microcentrifuge tubes and keep on ice for 30 min with occasional vortexing.</li>
		<li>Centrifuge the cell lysates at 13,000 x g for 10 min at 4 &#176;C.</li>
		<li>Collect the supernatant, aliquot 50 &#181;L of the supernatant into 1.5 mL microcentrifuge tubes, and store at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare the nuclear extracts using a nuclear extraction kit (see Table of Materials).
	<ol>
		<li>Gently resuspend the cell pellets in 500 &#181;L of lysis buffer NL supplemented with 1x protease inhibitor cocktail and 0.1 M dithiothreitol (DTT) by pipetting the cell pellets up and down several times.</li>
		<li>Add 25 &#181;L of detergent solution NP to the cell suspension and vortex for 10 s at maximum speed.</li>
		<li>Centrifuge at 10,000 x g for 5 min at 4 &#176;C.</li>
		<li>Collect the supernatant (cytoplasmic extracts), aliquot 50 &#181;L of the supernatant into 1.5 mL microcentrifuge tubes, and store at -80 &#176;C.</li>
		<li>Resuspend the pellet containing cell nuclei in 500 &#181;L of lysis buffer NL supplemented with 1x protease inhibitor cocktail and 0.1 M DTT by vortexing for 5 s at maximum speed.</li>
		<li>Centrifuge at 10,000 x g for 5 min at 4 &#176;C and save the nuclear pellet.</li>
		<li>Resuspend the nuclear pellet in 50 &#181;L of Extraction Buffer NX1 supplemented with 1x protease inhibitor cocktail by pipetting the pellet up and down several times.</li>
		<li>Incubate for 30 min on ice, vortexing for 10 s every 5 min at maximum speed.</li>
		<li>Centrifuge at 12,000 x g for 10 min at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Desalt the nuclear extracts.
	<ol>
		<li>Collect the supernatant from step 2.5.9 and transfer into the mini dialysis devices with a maximum volume of 100 &#181;L per unit.</li>
		<li>Cap the mini dialysis devices and place them in a flotation device.</li>
		<li>Put the flotation device in a beaker containing 500 mL of pre-chilled dialysis buffer (20mM Tris-HCl pH 7.4, 20% glycerol, 100mM potassium chloride (KCl), 0.2 mM ethylenediaminetetraacetic acid (EDTA), 0.2 mM phenylmethylsulfonyl fluoride (PMSF) and 0.5 mM DTT) and incubate for 30 min at 4 &#176;C with gentle stirring. 
		Caution: PMSF is hazardous. Avoid direct contact with skin or inhalation.</li>
		<li>Collect the samples from the corner of the mini dialysis devices and transfer into new 1.5 mL microcentrifuge tubes.</li>
		<li>Centrifuge at 12,000 x g for 10 min at 4 &#176;C, aliquot 25 &#181;L of each supernatant into a 1.5 mL microcentrifuge tube, and store at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Evaluation of the Hypoxia Treatment by Detection of the Protein Expression and Subcellular Localization of HIF-1&#945;
<ol>
	<li>Determine the protein concentration of the whole cell or nuclear/cytoplasmic extracts using the microplate assay of a bicinchoninic acid (BCA) protein assay kit according to manufacturer&#39;s instructions18.</li>
	<li>Dilute the cell lysates in 1x Laemmli sample buffer containing 5% 2-Mercaptoethanol and boil at 95 &#176;C for 5 min. 
	Caution: Do not touch the surface of the heating block, since it may cause burns.</li>
	<li>Separate the proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
	<ol>
		<li>Load equal amounts of protein (25 &#181;g) into each well of a SDS-PAGE precast gradient gels (4&#8211;20%), along with 3 &#181;L of the molecular weight marker.</li>
		<li>Run the gel in running buffer containing 2.5 mM Tris, 19.2 mM glycine, 0.01% SDS, PH8.3 for 30 min at 200 V.</li>
	</ol>
	</li>
	<li>Transfer the proteins to from the gel to the nitrocellulose membrane.
	<ol>
		<li>Assemble the transfer sandwich (filter paper-gel-membrane-filter paper) with the gel on the anode side and the membrane on the cathode side of the cassette.</li>
		<li>Place the cassette in the transfer tank filled with transfer buffer containing 2.5 mM trisaminomethane (Tris), 19.2 mM glycine and 20% methanol. 
		CAUTION: Methanol is flammable and should be stored inside the flammable liquid storage cabinet.</li>
		<li>Carry out the transfer for 1 h at 100 V in the cold room.</li>
	</ol>
	</li>
	<li>Block the blot in 10 mL blocking buffer containing 50 mM Tris-Cl (pH 7.6), 150 mM NaCl, 0.1 Tween 20 and 5% non-fat milk for 1 h at room temperature on a shaker.</li>
	<li>Incubate the blot with anti-HIF-1&#945; antibody (1/500 dilution) in the same blocking buffer overnight at 4 &#176;C.</li>
	<li>Wash the blot 3 times for 5 min each time with 50 mL TBS-T.</li>
	<li>Incubate the blot with horseradish peroxidase (HRP)-conjugated secondary antibody (1/1,000 dilution) in 10 mL TBS-T containing 5% non-fat dry milk for 1 h at room temperature.</li>
	<li>Wash the blot 3 times for 5 min each time with 50 mL TBS-T.</li>
	<li>Mix 500 &#181;L each of enhanced chemilumescent (ECL) reagent A and B in a 1.5 mL microcentrifuge tube and vortex briefly.</li>
	<li>Apply the ECL substrate to the blot and incubate for 1 min at room temperature.</li>
	<li>Capture the chemiluminescent signals using a charge-coupled device (CCD) camera-based imaging system.
	<ol>
		<li>Drain excess ECL substrate by touching the edge of the membrane with a tissue paper and place the membrane in a sheet protector.</li>
		<li>Place the membrane on the sample tray of the CCD camera-based imaging system.</li>
		<li>Launch image processing software (see Table of Materials) and capture the images with the following settings: File &#8594; New protocol &#8594; Single channel &#8594; Protocol Setup &#8594; Gel imaging (Application: Chemi; Imaging Area: Bio-Rad Ready gel; Imaging Exposure: The software will automatically optimize the exposure time for intense bands) &#8594; Run protocol 
		NOTE: The exposure time can be set manually to achieve the optimal images.</li>
	</ol>
	</li>
</ol>
4. Immunoprecipitation and Detection of the Immunoprecipitated Proteins
<ol>
	<li>Wash 50 &#181;L of protein A/G sepharose beads in 500 &#181;L of TBS buffer in 1.5 mL microcentrifuge tubes and pellet the beads by centrifugation at 3,000 x g for 2 min at 4 &#176;C.</li>
	<li>Discard the supernatant and resuspend the beads in 100 &#181;L of TBS buffer.</li>
	<li>Add 2 &#181;L of mouse monoclonal anti-ARNT antibody (1.4 mg/mL) or mouse Immunoglobulin G (IgG) (1.4 mg/mL) that was prepared by reconstituting 0.7 mg mouse IgG in 500 &#181;L TBS buffer.</li>
	<li>Place the 1.5 mL microcentrifuge tubes containing the beads in a tube rotator and incubate for 2 h at 10 rpm in the cold room. 
	NOTE: Handle the tubes gently and keep the suspension containing the beads at the bottom of the tube.</li>
	<li>Pellet the beads by centrifugation at 3,000 x g for 2 min at 4 &#176;C and discard the supernatants.</li>
	<li>Dilute 200 &#181;g of the nuclear protein lysate obtained in step 2.6.5 in 800 &#181;L of the IP buffer consisting of 50 mM Tris-HCl (pH 7.4), 180 mM NaCl, 20% glycerol, 0.2% NP-40 and 1x protease inhibitor cocktail. Incubate the lysate with the antibody-coupled beads obtained in step 4.5 overnight at 4 &#176;C.</li>
	<li>Pellet the beads by centrifugation at 3,000 x g for 2 min at 4 &#176;C, discard the supernatants and wash the beads 3 times with 1 mL ice-cold TBS containing 0.2% NP-40.</li>
	<li>Boil the beads in 50 &#181;L Laemmli sample buffer at 95 &#176;C for 5 min.</li>
	<li>Centrifuge the beads at 10,000 x g for 5 min at 4 &#176;C, collect the supernatant, and discard the beads. 
	NOTE: The supernatant can be stored at 4 &#176;C for the short term or -20 &#176;C for the long term.</li>
	<li>Detect the presence of HIF-1&#945; from the immunoprecipitated protein complexes by western blot as previously described in step 3.3 onwards. 
	NOTE: Protein quantification is not required for this step. Load full volume (50 &#181;L) of the supernatant of each sample into each well of a SDS-PAGE precast gradient gels (4&#8211;20%)</li>
</ol>

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The authors declare no conflicts of interest.

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><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1.0 M Tris-HCl Buffer, pH 7.4&amp;nbsp;</td><td>1st BASE</td><td>1415</td><td></td></tr><tr><td>Protein A/G Sepharose beads</td><td>Abcam</td><td>ab193262</td><td></td></tr><tr><td>Natural Mouse IgG protein</td><td>Abcam</td><td>ab198772</td><td></td></tr><tr><td>EDTA</td><td>Bio-Rad</td><td>1610729</td><td></td></tr><tr><td>2x Laemmli Sample Buffer</td><td>Bio-Rad</td><td>1610737</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Bio-Rad</td><td>1610710</td><td></td></tr><tr><td>Nitrocellulose Membrane &amp;nbsp;&amp;nbsp;</td><td>Bio-Rad</td><td>1620112</td><td></td></tr><tr><td>Blotting-Grade Blocker</td><td>Bio-Rad</td><td>1706404</td><td>Non-fat dry milk for western blotting applications</td></tr><tr><td>10x Tris Buffered Saline (TBS)</td><td>Bio-Rad</td><td>1706435</td><td></td></tr><tr><td>10% Tween 20</td><td>Bio-Rad</td><td>1610781</td><td></td></tr><tr><td>10x Tris/Glycine/SDS</td><td>Bio-Rad</td><td>1610732</td><td></td></tr><tr><td>10x Tris/Glycine Buffer&amp;nbsp;</td><td>Bio-Rad</td><td>1610771</td><td></td></tr><tr><td>Precision Plus Protein Dual Color Standards</td><td>Bio-Rad</td><td>1610374</td><td></td></tr><tr><td>Anti-rabbit IgG, HRP-linked Antibody</td><td>Cell Signaling</td><td>7074</td><td></td></tr><tr><td>Anti-mouse IgG, HRP-linked Antibody&amp;nbsp;</td><td>Cell Signaling</td><td>7076</td><td></td></tr><tr><td>SignalFire ECL Reagent</td><td>Cell Signaling</td><td>6883</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline</td><td>Corning</td><td>21-030-CV</td><td></td></tr><tr><td>Phenylmethylsulfonyl fluoride (PMSF)</td><td>Merck Millipore</td><td>52332</td><td></td></tr><tr><td>ARNT/HIF-1 beta Antibody&amp;nbsp;</td><td>Novus Biologicals</td><td>NB100-124&amp;nbsp;</td><td>Concentration: 1.4 mg/mL</td></tr><tr><td>HIF-1 alpha Antibody</td><td>Novus Biologicals</td><td>NB100-479</td><td>Concentration: 1.0 mg/mL</td></tr><tr><td>YY1 Antibody</td><td>Novus Biologicals</td><td>NBP1-46218</td><td>Concentration: 0.2 mg/mL</td></tr><tr><td>Qproteome Nuclear Protein Kit</td><td>Qiagen</td><td>37582</td><td>Lysis buffer NL and Extraction Buffer NX1 are provied in the kit</td></tr><tr><td>GAPDH Antibody</td><td>Santa Cruz</td><td>sc-47724</td><td>Concentration: 0.2 mg/mL</td></tr><tr><td>Glycerol (&amp;ge;99%)</td><td>Sigma</td><td>G5516</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>RIPA buffer</td><td>Sigma</td><td>R0278</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Sigma</td><td>71376</td><td></td></tr><tr><td>NP-40</td><td>Sigma</td><td>127087-87-0</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM, 4.5 g/L glucose)</td><td>Thermo Fisher Scientific</td><td>11995065</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Thermo Fisher Scientific</td><td>R0861</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fisher Scientific</td><td>10270106</td><td></td></tr><tr><td>HEK293A cell line</td><td>Thermo Fisher Scientific</td><td>R70507</td><td></td></tr><tr><td>Methanol&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>67-56-1</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Pierce Protease Inhibitor Tablets&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>88660</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Fisher Scientific</td><td>23225</td><td></td></tr><tr><td>QSP gel loading tip&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>QSP#010-R204-Q-PK</td><td>1-200 uL</td></tr><tr><td>&lt;strong&gt;Equipment/Instrument&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Thick Blot Filter Paper, Precut, 7.5 x 10 cm</td><td>Bio-Rad</td><td>1703932</td><td></td></tr><tr><td>Mini-PROTEAN&amp;nbsp;Tetra Vertical Electrophoresis Cell for Mini Precast Gels, with Mini Trans-Blot&amp;nbsp;Module and PowerPac Basic Power Supply</td><td>Bio-Rad</td><td>1658034</td><td></td></tr><tr><td>4&amp;ndash;15% Mini-PROTEAN TGX Precast Protein Gels</td><td>Bio-Rad</td><td>4561083</td><td></td></tr><tr><td>ChemiDoc XRS+ System</td><td>Bio-Rad</td><td>1708265</td><td></td></tr><tr><td>I-Glove</td><td>BioSpherix</td><td>I-Glove</td><td></td></tr><tr><td>Synergy HTX Multi-Mode Microplate Reader&amp;nbsp;</td><td>BioTek</td><td>BTS1LFTA</td><td></td></tr><tr><td>Costar 5mL Stripette Serological Pipets</td><td>Corning</td><td>4487</td><td></td></tr><tr><td>Costar 10mL Stripette Serological Pipets</td><td>Corning</td><td>4488</td><td></td></tr><tr><td>Costar 25mL Stripette Serological Pipets</td><td>Corning</td><td>4251</td><td></td></tr><tr><td>Corning 96-Well Clear Bottom Black Polystyrene Microplates</td><td>Corning</td><td>3631</td><td></td></tr><tr><td>15mL High Clarity PP conical Centrifuge Tubes</td><td>Corning</td><td>352095</td><td></td></tr><tr><td>Small Cell Scraper</td><td>Corning</td><td>3010</td><td></td></tr><tr><td>Gilson&amp;nbsp;Pipetman L 4-pipettes kit&amp;nbsp;</td><td>Gilson</td><td>F167370</td><td>P2, P20, P200, P1000 and accessories</td></tr><tr><td>1.5mL Polypropylene Microcentrifuge Tubes</td><td>Greiner Bio-One&amp;nbsp;</td><td>616201</td><td></td></tr><tr><td>PIPETBOY acu 2 Pipettor</td><td>INTEGRA Biosciences</td><td>155 000&amp;nbsp;</td><td></td></tr><tr><td>Justrite Flammable Liquid Storage Cabinets</td><td>Justrite Manufacturing Co.</td><td>896000</td><td></td></tr><tr><td>Vortex mixer</td><td>Labnet</td><td>S0200</td><td></td></tr><tr><td>CO2 incubator</td><td>NuAire</td><td>NU-5820</td><td></td></tr><tr><td>Orbital shakers</td><td>Stuart</td><td>SSL1</td><td></td></tr><tr><td>Tube rotator SB3</td><td>Stuart</td><td>SB3</td><td></td></tr><tr><td>MicroCL 21R Microcentrifuge</td><td>Thermo Fisher Scientific</td><td>75002470</td><td></td></tr><tr><td>Sorvall ST 16 Centrifuge</td><td>Thermo Fisher Scientific</td><td>75004240</td><td></td></tr><tr><td>Tissue Culture Dishes (100 mm)</td><td>Thermo Fisher Scientific</td><td>150350</td><td></td></tr><tr><td>Slide-A-Lyzer MINI Dialysis Device</td><td>Thermo Fisher Scientific</td><td>69580</td><td>10K MWCO, 0.1 mL</td></tr><tr><td>Float Buoys for 0.1mL Slide-A-Lyzer MINI Dialysis Devices</td><td>Thermo Fisher Scientific</td><td>69588</td><td></td></tr><tr><td>LSE Digital Dry Bath Heaters</td><td>Thermo Fisher Scientific</td><td>1168H25</td><td></td></tr><tr><td>Thermo Scientific&amp;nbsp;1300 Series A2 Class II, Type A2 Bio Safety Cabinets</td><td>Thermo Fisher Scientific</td><td>13-261-308</td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Image Lab Software</td><td>Bio-Rad</td><td>1709691</td><td></td></tr></tbody></table>

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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Research

JoVE Journal

Biology

17.8K Views.  Nanyang Technological University. Hier beschrijven we een co-immunoprecipitation protocol om te bestuderen van de eiwit-eiwit interactie tussen endogene nucleaire eiwitten hypoxische omstandigheden. Deze methode is geschikt voor de demonstratie van de interacties tussen transcriptiefactoren en transcriptionele co regelgevers op hypoxie.

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

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

Biochemistry

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which may be essential for a given protein&#39;s role both physiologically and pathologically. Enrichment of PTMs is often necessary when investigating the PTM status of a target protein, because PTMs are often transient and relatively low in abundance. Many pitfalls are encountered when enriching for a PTM of a target protein, such as buffer incompatibility, the target protein antibody is not IP-compatible, loss of PTM signal, and others. The degree of difficulty is magnified when investigating multiple PTMs like acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation for a given target protein. Studying a combination of these PTMs may be necessary, as crosstalk between PTMs is prevalent and critical for protein regulation. Often, these PTMs are studied in different lysis buffers and with unique inhibitor compositions. To simplify the process, a unique denaturing lysis system was developed that effectively isolates and preserves these four PTMs; thus, enabling investigation of potential crosstalk in a single lysis system. A unique filter system was engineered to remove contaminating genomic DNA from the lysate, which is a problematic by-product of denaturing buffers. Robust affinity matrices targeting each of the four PTMs were developed in concert with the buffer system to maximize the enrichment and detection of the endogenous states of these four PTMs. This comprehensive PTM detection toolset streamlines the process of obtaining critical information about whether a protein is modified by one or more of these PTMs.

Investigating multiple, endogenous post-translational modifications for a target protein can be extremely challenging. Techniques described here utilize an optimized lysis buffer and filter system developed with specific PTM-targeting, affinity matrices to detect the acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation modifications for a target protein using a single, streamlined system.

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

Genetics

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings.&#160;The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

Neuroscience

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Chromatin Immunoprecipitation Assay: A Technique to Study Protein-DNA Interactions in Cells

Source: Fuchs, A. R.,et al. <a href="https://www.jove.com/t/58270">A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia.</a> J. Vis. Exp. (2018).
This video describes protocol for chromatin immunoprecipitation (ChIP) assay, which is a classic technique to demonstrate protein-DNA interactions.This technique is used to identify target genes in human chronic lymphocytic leukemia cells.

Source: Fuchs, A. R.,et al. A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia. J. Vis. Exp. ...

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

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