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

Summary

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.

Abstract

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 α subunit (HIF-1α) and constitutively expressed β subunit (HIF-1β) 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-1α stability, hypoxia also triggers the nuclear translocation of many transcription factors including HIF-1α 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-1α 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.

Introduction

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 cancer^1,2. Hypoxia-inducible factors (HIFs) function as heterodimers composed of an oxygen-regulated α subunit and a constitutively expressed β subunit also known as ARNT³. Three isoforms of the HIF-α subunits (HIF-1α, HIF-2α and HIF-3α) and three HIF-β subunits (ARNT/HIF-1β, ARNT2 and ARNT3) have been identified to date. HIF-1α and ARNT are ubiquitously expressed, whereas HIF-2α, HIF-3α, 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-1α 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 "canonical" 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-κB)^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-1α protein levels are tightly regulated by cellular oxygen levels. At normoxia (21% oxygen), HIF-1α proteins are rapidly degraded^10,11. The short half-life of HIF-1α at normoxia presents specific technical challenges for the detection of the protein from cell extracts, as well as for the identification of HIF-1α-interacting proteins. Furthermore, several transcription factors including those of the HIF-1 complex translocate into the nucleus under hypoxic conditions^12,13,14. 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 modulation^15,16. 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-1α 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.

Protocol

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

1. Prepare four 10 cm dishes and seed 3–5 x 10⁶ HEK293A cells per dish in 10 mL Dulbecco's modified Eagle'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 °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.
2. 24 h after seeding, when the cells have reached 80–90% confluency, put two dishes into the hypoxia subchamber in the incubator glovebox (see Table of Materials) with 1% O₂ and 5% CO₂ at 37 °C, and keep the other two dishes at normoxia (21% O₂, 5% CO₂ at 37 °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 0–5%, and the duration of the hypoxia treatment can be varied depending on the cell type and study objective.

2. Whole Cell and Nuclear Extraction

NOTE: See Table 1 for information on buffers used in this protocol.

1. Harvest the normoxia control cells.
   1. Remove the culture media by aspiration and rinse the cells with 10 mL DPBS (PH 7.0–7.2) 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.
   2. 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.
   3. Transfer the cell suspension into 15 mL conical tubes and keep on ice.
2. Harvest the cells cultured under hypoxic conditions.
   1. 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 °C) for 24 h in advance.
   2. 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.
   3. 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.
   4. 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.
   5. Add 5 mL ice-cold pre-equilibrated DPBS with a 5 mL pipette and dislodge the cells by scraping with a cell scraper.
   6. 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.
   7. 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.
3. 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 °C.
4. Prepare the whole cell extracts.
   1. Resuspend the cell pellets in 500 µL 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.
   2. Transfer the cell lysates into new 1.5 mL microcentrifuge tubes and keep on ice for 30 min with occasional vortexing.
   3. Centrifuge the cell lysates at 13,000 x g for 10 min at 4 °C.
   4. Collect the supernatant, aliquot 50 µL of the supernatant into 1.5 mL microcentrifuge tubes, and store at -80 °C.
5. Prepare the nuclear extracts using a nuclear extraction kit (see Table of Materials).
   1. Gently resuspend the cell pellets in 500 µL 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.
   2. Add 25 µL of detergent solution NP to the cell suspension and vortex for 10 s at maximum speed.
   3. Centrifuge at 10,000 x g for 5 min at 4 °C.
   4. Collect the supernatant (cytoplasmic extracts), aliquot 50 µL of the supernatant into 1.5 mL microcentrifuge tubes, and store at -80 °C.
   5. Resuspend the pellet containing cell nuclei in 500 µL of lysis buffer NL supplemented with 1x protease inhibitor cocktail and 0.1 M DTT by vortexing for 5 s at maximum speed.
   6. Centrifuge at 10,000 x g for 5 min at 4 °C and save the nuclear pellet.
   7. Resuspend the nuclear pellet in 50 µL of Extraction Buffer NX1 supplemented with 1x protease inhibitor cocktail by pipetting the pellet up and down several times.
   8. Incubate for 30 min on ice, vortexing for 10 s every 5 min at maximum speed.
   9. Centrifuge at 12,000 x g for 10 min at 4 °C.
6. Desalt the nuclear extracts.
   1. Collect the supernatant from step 2.5.9 and transfer into the mini dialysis devices with a maximum volume of 100 µL per unit.
   2. Cap the mini dialysis devices and place them in a flotation device.
   3. 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 °C with gentle stirring.
      Caution: PMSF is hazardous. Avoid direct contact with skin or inhalation.
   4. Collect the samples from the corner of the mini dialysis devices and transfer into new 1.5 mL microcentrifuge tubes.
   5. Centrifuge at 12,000 x g for 10 min at 4 °C, aliquot 25 µL of each supernatant into a 1.5 mL microcentrifuge tube, and store at -80 °C.

3. Evaluation of the Hypoxia Treatment by Detection of the Protein Expression and Subcellular Localization of HIF-1α

1. 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's instructions¹⁸.
2. Dilute the cell lysates in 1x Laemmli sample buffer containing 5% 2-Mercaptoethanol and boil at 95 °C for 5 min.
   Caution: Do not touch the surface of the heating block, since it may cause burns.
3. Separate the proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
   1. Load equal amounts of protein (25 µg) into each well of a SDS-PAGE precast gradient gels (4–20%), along with 3 µL of the molecular weight marker.
   2. 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.
4. Transfer the proteins to from the gel to the nitrocellulose membrane.
   1. 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.
   2. 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.
   3. Carry out the transfer for 1 h at 100 V in the cold room.
5. 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.
6. Incubate the blot with anti-HIF-1α antibody (1/500 dilution) in the same blocking buffer overnight at 4 °C.
7. Wash the blot 3 times for 5 min each time with 50 mL TBS-T.
8. 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.
9. Wash the blot 3 times for 5 min each time with 50 mL TBS-T.
10. Mix 500 µL each of enhanced chemilumescent (ECL) reagent A and B in a 1.5 mL microcentrifuge tube and vortex briefly.
11. Apply the ECL substrate to the blot and incubate for 1 min at room temperature.
12. Capture the chemiluminescent signals using a charge-coupled device (CCD) camera-based imaging system.
    1. Drain excess ECL substrate by touching the edge of the membrane with a tissue paper and place the membrane in a sheet protector.
    2. Place the membrane on the sample tray of the CCD camera-based imaging system.
    3. Launch image processing software (see Table of Materials) and capture the images with the following settings: File → New protocol → Single channel → Protocol Setup → Gel imaging (Application: Chemi; Imaging Area: Bio-Rad Ready gel; Imaging Exposure: The software will automatically optimize the exposure time for intense bands) → Run protocol
       NOTE: The exposure time can be set manually to achieve the optimal images.

4. Immunoprecipitation and Detection of the Immunoprecipitated Proteins

1. Wash 50 µL of protein A/G sepharose beads in 500 µL 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 °C.
2. Discard the supernatant and resuspend the beads in 100 µL of TBS buffer.
3. Add 2 µL 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 µL TBS buffer.
4. 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.
5. Pellet the beads by centrifugation at 3,000 x g for 2 min at 4 °C and discard the supernatants.
6. Dilute 200 µg of the nuclear protein lysate obtained in step 2.6.5 in 800 µL 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 °C.
7. Pellet the beads by centrifugation at 3,000 x g for 2 min at 4 °C, discard the supernatants and wash the beads 3 times with 1 mL ice-cold TBS containing 0.2% NP-40.
8. Boil the beads in 50 µL Laemmli sample buffer at 95 °C for 5 min.
9. Centrifuge the beads at 10,000 x g for 5 min at 4 °C, collect the supernatant, and discard the beads.
   NOTE: The supernatant can be stored at 4 °C for the short term or -20 °C for the long term.
10. Detect the presence of HIF-1α 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 µL) of the supernatant of each sample into each well of a SDS-PAGE precast gradient gels (4–20%)

Results

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-1α and ARNT protein levels were examined in whole cell or nuclear/cytoplasmic extracts by western blot. As expected, total HIF-1α levels were upregulated by hypoxia, whereas ARNT levels in total cellular lysates were n...

Discussion

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 ...

Materials

List of materials used in this article

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