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. 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.
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 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 degraded10,11. 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 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-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.
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
2. Whole Cell and Nuclear Extraction
NOTE: See Table 1 for information on buffers used in this protocol.
3. Evaluation of the Hypoxia Treatment by Detection of the Protein Expression and Subcellular Localization of HIF-1α
4. Immunoprecipitation and Detection of the Immunoprecipitated Proteins
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 not significantly altered (Figure 1A). In addition, hypoxia induced nuclear accumulation of both HIF-1α and ARNT in HEK293A cells (Figure 1B), which is consistent with previous reports, although cytoplasmic expression of ARNT was not detected in some of the tested cell lines19.
Next, we demonstrated the interaction between HIF-1α and ARNT following hypoxia treatment. Nuclear extracts were prepared from HEK293A cells exposed to normoxic or hypoxic conditions for 4 h. Co-immunoprecipitation experiments were performed using nuclear extracts from HEK293A cells. As shown in Figure 2, HIF-1α was co-immunoprecipitated together with ARNT from the nuclear extracts of hypoxic HEK293A cells.
Taken together, the protocol described can be successfully used to induce a hypoxic response in cells and to further determine physiological protein binding of endogenously expressed HIF-1α/ARNT complexes within the nucleus.
Figure 1: Regulation of protein expression and subcellular localization of HIF-1 components by hypoxia. (A) Immunoblot analysis of total HIF-1α or ARNT expression in HEK293A cells. The cells were exposed to hypoxia for 4 h (H) or kept at normoxia (N). Proteins from whole-cell extracts were separated by SDS-PAGE and analyzed by immunoblotting with anti-HIF-1α, anti-ARNT and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies. GAPDH was used as a loading control. (B) Immunoblot analysis of HIF-1α and ARNT expression in subcellular fractions of HEK293A cells. HEK293A cells were cultured under hypoxic conditions for 4 h (H) or kept at normoxia (N). 25 µg of protein from nuclear or cytoplasmic extracts were analyzed by immunoblotting using anti-HIF-1α, anti-ARNT, anti-yin and yang 1 (YY1) and anti-GAPDH antibodies. YY1 and GAPDH were used as loading controls for nuclear and cytoplasmic fractions, respectively. Please click here to view a larger version of this figure.
Figure 2: HIF-1α interacts with ARNT under hypoxic conditions. HEK293A cells were exposed to hypoxia for 4 h or kept at normoxia as controls. Nuclear extracts from HEK293A cells were prepared and immunoprecipitations were performed using an anti-ARNT antibody. Nuclear extracts (inputs) and immunoprecipitated proteins were analyzed by immunoblotting using anti-HIF-1α, anti-ARNT and anti-YY1 antibodies. YY1 was used as a loading control for the inputs while IgG was used as the negative control for the co-immunoprecipitation experiments. Please click here to view a larger version of this figure.
Solution | Components | Comments |
Dialysis buffer | 20 mM Tris-HCl (pH 7.4), 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.2 mM PMSF and 0.5 mM DTT | Add PMSF and DTT immediately before use. |
Running buffer | 2.5 mM Tris-HCl (PH 8.3), 19.2 mM glycine and 0.01% SDS | Mix 100 mL 10x Tris/Glycine/SDS buffer with 900 mL ddH2O. |
Transfer buffer | 2.5 mM Tris-HCl (PH 8.3), 19.2 mM glycine and 20% methanol | Mix 100 mL 10x Tris/Glycine buffer with 200 mL methanol and 700 mL ddH2O. |
TBS buffer | 50 mM Tris-Cl (pH 7.6) and 150 mM NaCl | Mix 100 mL 10x TBS buffer with 900 mL ddH2O. |
TBS-T buffer | 50 mM Tris-Cl (pH 7.6), 150 mM NaCl and 0.1% Tween 20 | Mix 100 mL 10x TBS buffer with 900 mL ddH2O and 1 mL of Tween 20. |
Blocking buffer | 50 mM Tris-Cl (pH 7.6), 150 mM NaCl, 0.1% Tween 20 and 5% non-fat milk | Dissolve 5 g of Blotting-Grade Blocker in 100 mL TBS-T buffer. |
IP buffer | 50 mM Tris-HCl (pH 7.4), 180 mM NaCl, 20% glycerol, 0.2% NP-40 and 1x protease inhibitor cocktail | Add protease inhibitor cocktail immediately before use. |
Table 1: Solution preparation.
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-1α is a highly unstable protein and it becomes rapidly degraded during re-oxygenation11. In addition, hypoxia triggers nuclear translocation of HIF-1 components in most mammalian cell lines. Therefore, conventional co-immunoprecipitation protocols have to be optimized for the identification of physiologically relevant HIF-1 interacting proteins following hypoxia treatment. Here, we describe a modified co-immunoprecipitation protocol that is used to demonstrate the interaction between HIF-1α and ARNT at hypoxia.
To avoid the degradation of HIF-1α during re-oxygenation, the hypoxic cells were harvested inside the processing chamber of the incubator glovebox which was pre-equilibrated to hypoxic conditions. The DPBS wash buffer was also equilibrated to hypoxic conditions before usage. If there is no hypoxia workstation available for processing, the hypoxic cells can be washed with pre-equilibrated hypoxic DPBS and thereafter harvested quickly on ice. Considering that hypoxia can trigger the nuclear translocation of HIF-1 components and that protein overexpression may induce artefactual results, endogenous nuclear proteins were used in this protocol. The use of nuclear extracts in the co-immunoprecipitation protocol has also been reported by others20. It represents a technical advantage for the study of interactions between nuclear proteins, because it minimizes the chance of nuclear proteins from being diluted out by whole-cell proteins. Furthermore, the use of nuclear extracts may be helpful for the reduction of the non-specific interactions, especially from highly abundant irrelevant cytoplasmic proteins, and for the improvement of nuclear protein stability by minimizing exposure to proteases present in the cytoplasm. The use of endogenous nuclear extracts instead of whole cell extracts for the co-immunoprecipitation shows a significant technical improvement, as we can only detect the interaction between HIF-1α and GABPα using endogenous nuclear extracts but not using whole cell extracts17.
In the described protocol, several conditions can be further optimized to improve results. We induced hypoxia in HEK293A cells by treating them with 1% oxygen for 4 h. However the oxygen levels and the duration of the hypoxia treatment can be varied for different cell types and adjusted according to the aims of the study. For instance, HIF-1α and HIF-2α can be differentially regulated by hypoxia in a cell type specific manner, indicating their distinct roles in different biological contexts. It has been shown that in neuroblastoma cells, HIF-1α is most active during short periods of intense hypoxia (1% O2), whereas HIF-2α is also active under mild hypoxia (5% O2) and becomes more active following chronic hypoxia treatment (48–72 h of hypoxic exposure)21. 0–5% O2 levels are commonly used for in vitro hypoxia treatments, where 1–5% O2, 0.1–1% O2 and 0–0.1% O2 are frequently defined as mild hypoxia, hypoxia and anoxia, respectively22. Salt concentration is another parameter that requires optimization, especially since it plays a critical role for ionic interactions in PPIs23. Nuclear extracts were used in this study with the nuclear proteins usually extracted using a buffer containing high concentrations of salt. Therefore, the nuclear extracts may have to be desalted prior to the co-immunoprecipitation experiments. Here, we desalted the nuclear extracts using a dialysis buffer containing 100 mM KCl, and mixed the dialyzed extracts with the IP buffer containing 180 mM NaCl proportionally to achieve a final salt concentration close to 150 mM, which is similar to physiological intracellular PPIs conditions. The final salt concentration can be modified depending on the properties of the PPIs of interest. In this protocol, GAPDH was used as a loading control for whole cell extracts and cytoplasmic fractions. We did not observe any significant hypoxia-induced regulatory effect on GAPDH expression in HEK293A cells. However, GAPDH has previously been shown to be upregulated by hypoxia in certain cell types24. With this in mind, one may need to use alternative proper loading controls when necessary25. Separately, we observed a significant level of background signal stemming from either the beads or antibodies used in the current protocol. To reduce the background, one can prolong the washing steps (10–15 min) or perform more than 3 washes (5–10 times). Alternatively, the ionic strength of the wash buffer can also be increased by titrating the salt concentration from 150 to 500 mM during washes. The lysates can also be pre-cleared by incubating with protein A/G sepharose beads for 1 h at 4 °C with rotation.
This protocol is limited to nuclear extracts and as such may not be suitable for the study of PPIs within other cellular compartments such as mitochondria and the endoplasmic reticulum. Similar to other conventional co-immunoprecipitation protocols, this method cannot be used to study PPIs in real-time or to determine if the PPIs are direct or indirect.
In summary, we provide a modified co-immunoprecipitation protocol for the identification of novel physiologically relevant HIF-1 interacting proteins. This protocol is also suitable for the study of the interactions between transcription factors and transcriptional co-regulators under hypoxic conditions. Although this protocol is designed specifically for the co-immunoprecipitation experiments under hypoxia conditions, a part of the described protocol for the normoxia control cells can also be used to study the nuclear PPIs under normoxia conditions.
The authors have nothing to disclose.
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.
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 |