Visualisering av Protein-protein Interaksjon i Kjerne- og cytoplasmatiske Fraksjoner av Co-immunoprecipitation og<em> In Situ</em> Nærhet hemorroider Assay
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
Nukleær faktor 90 (NF90) er et multi-isoform-protein med en rekke funksjoner, inkludert respons på virusinfeksjon, for regulering av interleukin-2 post-transkripsjon og regulering av miRNA biogenese 1-3. RBM3 er en RNA-bindende protein, er involvert i oversettelse og miRNA biogenesis og kan være forårsaket av ulike stressfaktorer inkludert hypotermi og hypoksi 4-6. Nylig fant vi NF90 og RBM3 i et proteinkompleks 7. Samspillet mellom NF90 og RBM3 er viktig å modulere protein kinase RNA-lignende endoplasmatiske retikulum kinase (PERK) aktivitet i utfoldet protein respons 7. Både NF90 og RBM3 ligger hovedsakelig i kjernen, men en liten andel av NF90 og RBM3 transport til cytoplasma og binde det til hverandre for bestemte funksjoner, for eksempel for å regulere PERK aktivitet. Derfor er det viktig å visualisere fordelingen av NF90-RBM3 interaksjoner i den subcellulære rommet, noe som kan indikeresine ulike roller i respektive rom.
For flere tiår siden, ble gjær to hybrid (Y2H) utviklet for å oppdage samspillet mellom to proteiner 8. Men på grunn av kunstig konstruksjon av fusjonerte proteiner, er falske positive resultater begrenset anvendelsen av denne metoden. I lang tid, co-immunutfelling ble de viktigste teknikk for å analysere protein-protein interaksjoner, spesielt i endogene forhold 9. For å analysere den ko-immunopresipitert proteinkompleks, er Western blot det mest hensiktsmessig teknikk, mens massespektrometri er brukt når super følsomhet og nøyaktighet er ønskelig. I de senere år har nærhet ringsanalyse blitt utviklet som en ny metode for å påvise protein-protein interaksjoner i begge celler og vev in situ 10,11.
Her sammenlignet vi det mest populære ko-immunoutfelling fremgangsmåte og en forholdsvis ny nærhet ligering analysemetoden i å fange NF90-RBM3 samhandling i subcellulære fraksjoner. Vi diskuterte også fordeler og begrensninger av begge teknikkene.
Protocol
Representative Results
Discussion
Det er flere fordeler så vel som ulemper for begge metodene. Som en relativt ny teknikk, en åpenbar fordel med nærhet ligation analysen er muligheten til å belyse protein-protein interaksjoner på enkeltcellenivå i stedet for en gruppe med heterogene celler. Bilder med høy styrke og oppløsning (for eksempel ved konfokal mikroskop) gi mulighet for kvantifisering ved å telle enkelt fluorescerende flekker. I motsetning til dette, kan den konvensjonelle kombinasjon av ko-immunoutfelling teknikken med Wester…
Divulgaciones
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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