共免疫沈降により核および細胞質画分中のタンパク質間相互作用の可視化と<em>その場で</em>近接ライゲーションアッセイ
Summary
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.
Abstract
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.
Introduction
核因子90（NF90）は、ウイルス感染に応答して、miRNAの生合成1-3のインターロイキン2転写後規制の規制を含む多数の機能を備えたマルチアイソフォームタンパク質です。 RBM3は、RNA結合タンパク質、翻訳およびmiRNA生合成に関与し、低体温症および低酸素4-6を含む様々なストレスによって誘導することができるです。最近、我々は、タンパク質複合体7にNF90とRBM3を発見しました。 NF90とRBM3の相互作用は、小胞体ストレス応答7にプロテインキナーゼRNA様小胞体キナーゼ（PERK）活性を調節することが不可欠です。 NF90とRBM3の両方がPERK活性を調節するために、例えば 、核内に主に位置するが、NF90とRBM3シャトル少量の細胞質へと特定の機能のためにお互いにそこにバインドされています。したがって、を示すことができる、細胞内区画にNF90-RBM3相互作用の分布を可視化することが重要ですそれぞれの区画での様々な役割。
数十年前、酵母2ハイブリッド（Y2H）は、2つのタンパク質8の間の相互作用を検出するために開発されました。しかし、融合タンパク質の人工的な構造のため、偽陽性の結果は、この方法の適用が制限されています。長い間、免疫共沈降は、特に内因性条件9に、タンパク質-タンパク質相互作用を分析するための主な手法でした。超感度および精度が所望される場合、質量分析法を用いているが、共免疫沈降したタンパク質複合体を分析するために、ウェスタンブロットは、最も便利な技術です。近年では、近接ライゲーションアッセイは、in situ 10,11 の両方の細胞および組織におけるタンパク質-タンパク質相互作用を検出するための新規な方法として開発されました。
ここでは、最も人気の共免疫沈降法とNF9を取り込むには比較的小説近接ライゲーションアッセイ法を比較しました細胞内画分中の0-RBM3相互作用。また、両方の技術の利点と限界を議論しました。
Protocol
Representative Results
Discussion
両方の方法にはいくつかの利点だけでなく、欠点があります。比較的新しい技術として、近接ライゲーションアッセイの明らかな利点は、単一細胞レベルの代わりに、異種の細胞のバッチでのタンパク質 – タンパク質相互作用を解明する可能性があります。高い大きさと（共焦点顕微鏡によって）解像度の画像は、単一の蛍光スポットを計数することによって定量化のための可能性を?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This study was supported by the Swiss National Science Foundation (SNSF, 31003A_163305).
Materials
| Dulbecco's Modified Eagle’s Medium (DMEM) | Sigma | D6429 | High glucose 4500 mg/L |
| Fetal bovine serum (FBS) | Gibco, Thermo Fisher Scientific | 10270106 | |
| Penicillin-Streptomycin (PenStrep) | BioConcept | 4-01F00-H | |
| NE-PER Nuclear and Cytoplasmic Extraction Reagents | Thermo Fisher Scientific | 78833 | |
| 1,4-Dithiothreitol (DTT) | Carl Roth | 6908.3 | |
| Dynabeads Protein G | Novex, Thermo Fisher Scientific | 10003D | |
| DRBP76 (NF90/NF110) antibody | BD Transduction Laboratories | 612154 | use 1:1000 for WB and 1:100 for ICC/PLA |
| RBM3 antibody | ProteinTech | 14363-1-AP | use 1:1000 for WB and 1:100 for ICC/PLA |
| Lamin A/C antibody | Cell Signaling Technology | #2032 | use 1:1000 for WB |
| anti-GAPDH antibody | Abcam | ab8245 | use 1:1000 for WB |
| normal rabbit IgG | Santa Cruz | sc-2027 | |
| anti-rabbit IgG, HRP-lined secodary antiboy | Cell Signaling Technology | #7074 | use 1:5000 for WB |
| anti-mouse HRP secondary antibody | Carl Roth | 4759.1 | use 1:5000 for WB |
| Clarity Western ECL Blotting Substrate | Bio-Rad | #1705060 | |
| NuPAGE Novex 4-12% Bis-Tris Gel | Novex, Thermo Fisher Scientific | NP0321BOX | |
| NuPAGE LDS Sample Buffer (4x) | Novex, Thermo Fisher Scientific | NP0007 | |
| 1,4-Dithiothreitol (DTT) | CarlRoth | 6908.1 | |
| NuPAGE MES SDS Running Buffer (20x) | Novex, Thermo Fisher Scientific | NP0002 | |
| NuPAGE Transfer Buffer (20x) | Novex, Thermo Fisher Scientific | NP00061 | |
| Amersham Hypond P 0.2 PVDF membrane | GE Healthcare Life Sciences | 10600021 | |
| Super RX X-ray film | Fujifilm | 4741029230 | |
| Poly-D-Lysine 8 Well Culture Slide | Corning BioCoat | 354632 | |
| Paraformaldehyde (PFA) | Sigma | P6148 | |
| Normal goat serum (NGS) | Gibco, Thermo Fisher Scientific | PCN5000 | |
| Goat anti-mouse IgG (H+L Antibody), Alexa Fluor 488 conjugate | Thermo Fisher Scientific | A-11001 | |
| Goat anti-rabbit IgG (H+L Antibody), Alexa Fluor 568 conjugate | Thermo Fisher Scientific | A-11011 | |
| 4′, 6-Diamidin-2-phenylindol (DAPI) | Sigma | D9542 | |
| Duolink PLA probe Anti-mouse PLUS | Sigma | DUO92001 | |
| Duolink PLA probe Anti-rabbit MINUS | Sigma | DUO92005 | |
| Duolink Detection Reagents Red | Sigma | DUO92008 | |
| Duolink Wash Buffers Fluorescence | Sigma | DUO82049 | |
| Duolink Mounting Medium with DAPI | Sigma | DUO82040 | |
| Mowiol 4-88 | Sigma | 81381 | |
| Microscope | Olympus | AX-70 | |
| CCD camera | SPOT | Insight 2MP Firewire | |
| X-ray film | Fujifilm | Super RX | |
| Film processing machine | Fujifilm | FPM-100A |
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