Name: visualization of protein-protein interaction in nuclear and cytoplasmic fractions by co-immunoprecipitation and in situ proximity ligation assay
Uploaded: 2017-01-16T15:00:00
Description: Watch this Scientific Journal Video about Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay at JoVE.com

This study was supported by the Swiss National Science Foundation (SNSF, 31003A_163305).

1. Co-immunoprecipitation
<ol>
	<li>Seed HEK293 cells at 2 x 105 cells per well in one 6-well plate in 2 ml Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) supplement with 10% fetal bovine serum (FBS) and 100 U/mL Penicillin-Streptomycin (Pen-Strep).</li>
	<li>Grow cells for 48 hr at 37 &#176;C with 5% CO2.</li>
	<li>Wash cells with cold phosphate-buffered saline (PBS) three times. Harvest cells by centrifugation at 500 x g for 5 min at 4 &#176;C.</li>
	<li>Prepare nuclear and cytoplasmic fracti...

<ol>
	<li>Pati&#241;o, C., Haenni, A. L., Urcuqui-Inchima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF90+isoforms,+a+new+family+of+cellular+proteins+involved+in+viral+replication?.">NF90 isoforms, a new family of cellular proteins involved in viral replication?.</a> Biochimie. 108, 20-24 (2015).</li><li>Shim, J., Lim, H., R Yates, J., Karin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+export+of+NF90+is+required+for+interleukin-2+mRNA+stabilization.">Nuclear export of NF90 is required for interleukin-2 mRNA stabilization.</a> Mol Cell. 10 (6), 1331-1344 (2002).</li><li>Sakamoto, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NF90-NF45+complex+functions+as+a+negative+regulator+in+the+microRNA+processing+pathway.">The NF90-NF45 complex functions as a negative regulator in the microRNA processing pathway.</a> Mol Cell Biol. 29 (13), 3754-3769 (2009).</li><li>Dresios, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold+stress-induced+protein+Rbm3+binds+60S+ribosomal+subunits,+alters+microRNA+levels,+and+enhances+global+protein+synthesis.">Cold stress-induced protein Rbm3 binds 60S ribosomal subunits, alters microRNA levels, and enhances global protein synthesis.</a> Proc Natl Acad Sci. 102 (6), 1865-1870 (2005).</li><li>Danno, S., Itoh, K., Matsuda, T., Fujita, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+expression+of+mouse+Rbm3,+a+cold-shock+protein,+in+Sertoli+cells+of+cryptorchid+testis.">Decreased expression of mouse Rbm3, a cold-shock protein, in Sertoli cells of cryptorchid testis.</a> Am J Pathol. 156 (5), 1685-1692 (2000).</li><li>Wellmann, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen-regulated+expression+of+the+RNA-binding+proteins+RBM3+and+CIRP+by+a+HIF-1-independent+mechanism.">Oxygen-regulated expression of the RNA-binding proteins RBM3 and CIRP by a HIF-1-independent mechanism.</a> J Cell Sci. 117 (Pt 9), 1785-1794 (2004).</li><li>Zhu, X., Zelmer, A., Kapfhammer, J. P., Wellmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold-inducible+RBM3+inhibits+PERK+phosphorylation+through+cooperation+with+NF90+to+protect+cells+from+endoplasmic+reticulum+stress.">Cold-inducible RBM3 inhibits PERK phosphorylation through cooperation with NF90 to protect cells from endoplasmic reticulum stress.</a> FASEB J. 30 (2), 624-634 (2016).</li><li>Fields, S., Song, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+genetic+system+to+detect+protein-protein+interactions.">A novel genetic system to detect protein-protein interactions.</a> Nature. 340 (6230), 245-246 (1989).</li><li>Verhelst, J., De Vlieger, D., Saelens, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-immunoprecipitation+of+the+Mouse+Mx1+Protein+with+the+Influenza+A+Virus+Nucleoprotein.">Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein.</a> J Vis Exp. (98), (2015).</li><li>S&#246;derberg, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observation+of+individual+endogenous+protein+complexes+in+situ+by+proximity+ligation.">Direct observation of individual endogenous protein complexes in situ by proximity ligation.</a> Nat Methods. 3 (12), 995-1000 (2007).</li><li>Jarvius, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+detection+of+phosphorylated+platelet-derived+growth+factor+receptor+beta+using+a+generalized+proximity+ligation+method.">In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method.</a> Mol Cell Proteomics. 6 (9), 1500-1509 (2007).</li><li>Liu, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+protein-protein+interactions+in+cross-talk+pathways+reveals+CRKL+protein+as+a+novel+prognostic+marker+in+hepatocellular+carcinoma.">Analysis of protein-protein interactions in cross-talk pathways reveals CRKL protein as a novel prognostic marker in hepatocellular carcinoma.</a> Mol Cell Proteomics. 12 (5), 1335-1349 (2013).</li></ol>

There are several benefits as well as shortcomings for both methods. As a relatively novel technique, an obvious advantage of proximity ligation assay is the feasibility to elucidate protein-protein interactions at single-cell level instead of a batch of heterogeneous cells. Images with high magnitude and resolution (e.g. by confocal microscope) provide the possibility for quantification by counting single fluorescent spots. In contrast, the conventional combination of co-immunoprecipitation technique with Weste...

Nuclear factor 90 (NF90) is a multi-isoform protein with numerous functions including the response to viral infection, regulation of interleukin-2 post-transcription and regulation of miRNA biogenesis 1-3. RBM3 is an RNA-binding protein, involved in translation and miRNA biogenesis and can be induced by various stressors including hypothermia and hypoxia 4-6. Recently, we found NF90 and RBM3 in a protein complex 7. The interaction of NF90 and RBM3 is essential to modulate protein kinase RNA-like endoplasmic reticulum kinase (PERK) activity in unfolded protein response 7. Both NF90 and RBM3 are located predominantly in the nucleus but a small proportion of NF90 and RBM3 shuttle into the cytoplasm and bind there to each other for specific functions, e.g. to regulate PERK activity. Therefore, it is important to visualize the distribution of NF90-RBM3 interactions in the subcellular compartment, which may indicate their various roles in respective compartment.
Decades ago, yeast two hybrid (Y2H) was developed to detect the interaction between two proteins 8. However, due to artificial construction of fused proteins, false positive results have restricted the application of this method. For a long time, co-immunoprecipitation was the main technique to analyze protein-protein interactions, especially in endogeneous conditions 9. To analyze the co-immunoprecipitated protein complex, Western blot is the most convenient technique, while mass spectrometry is used when super sensitivity and accuracy are desired. In recent years, proximity ligation assay has been developed as a novel method to detect protein-protein interactions in both cells and tissues in situ&#160;10,11.
Here, we compared the most popular co-immunoprecipitation method and relatively novel proximity ligation assay method in capturing NF90-RBM3 interaction in subcellular fractions. We also discussed the advantages and limitations of both techniques.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td>Sigma</td>
			<td>D6429</td>
			<td>High glucose 
			4500 mg/L</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco, Thermo Fisher Scientific</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (PenStrep)</td>
			<td>BioConcept</td>
			<td>4-01F00-H</td>
			<td></td>
		</tr>
		<tr>
			<td>NE-PER Nuclear and Cytoplasmic Extraction Reagents</td>
			<td>Thermo Fisher Scientific</td>
			<td>78833</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-Dithiothreitol (DTT)</td>
			<td>Carl Roth</td>
			<td>6908.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads Protein G</td>
			<td>Novex, Thermo Fisher Scientific</td>
			<td>10003D</td>
			<td></td>
		</tr>
		<tr>
			<td>DRBP76 (NF90/NF110) antibody</td>
			<td>BD Transduction Laboratories</td>
			<td>612154</td>
			<td>use 1:1000 for WB and 1:100 for ICC/PLA</td>
		</tr>
		<tr>
			<td>RBM3 antibody</td>
			<td>ProteinTech</td>
			<td>14363-1-AP</td>
			<td>use 1:1000 for WB and 1:100 for ICC/PLA</td>
		</tr>
		<tr>
			<td>Lamin A/C antibody</td>
			<td>Cell Signaling Technology</td>
			<td>#2032</td>
			<td>use 1:1000 for WB&#160;</td>
		</tr>
		<tr>
			<td>anti-GAPDH antibody</td>
			<td>Abcam</td>
			<td>ab8245</td>
			<td>use 1:1000 for WB&#160;</td>
		</tr>
		<tr>
			<td>normal rabbit IgG</td>
			<td>Santa Cruz</td>
			<td>sc-2027</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rabbit IgG, HRP-lined secodary antiboy</td>
			<td>Cell Signaling Technology</td>
			<td>#7074</td>
			<td>use 1:5000 for WB&#160;</td>
		</tr>
		<tr>
			<td>anti-mouse HRP secondary antibody</td>
			<td>Carl Roth</td>
			<td>4759.1</td>
			<td>use 1:5000 for WB&#160;</td>
		</tr>
		<tr>
			<td>Clarity Western ECL Blotting Substrate</td>
			<td>Bio-Rad</td>
			<td>#1705060</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE Novex 4-12% Bis-Tris Gel</td>
			<td>Novex, Thermo Fisher Scientific</td>
			<td>NP0321BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE LDS Sample Buffer (4x)</td>
			<td>Novex, Thermo Fisher Scientific</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-Dithiothreitol (DTT)</td>
			<td>CarlRoth</td>
			<td>6908.1</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE MES SDS Running Buffer (20x)</td>
			<td>Novex, Thermo Fisher Scientific</td>
			<td>NP0002</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE Transfer Buffer (20x)</td>
			<td>Novex, Thermo Fisher Scientific</td>
			<td>NP00061</td>
			<td></td>
		</tr>
		<tr>
			<td>Amersham Hypond P 0.2 PVDF membrane</td>
			<td>GE Healthcare Life Sciences</td>
			<td>10600021</td>
			<td></td>
		</tr>
		<tr>
			<td>Super RX X-ray film</td>
			<td>Fujifilm</td>
			<td>4741029230</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine 8 Well Culture Slide</td>
			<td>Corning BioCoat</td>
			<td>354632</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum (NGS)</td>
			<td>Gibco, Thermo Fisher Scientific</td>
			<td>PCN5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG (H+L Antibody), Alexa Fluor 488 conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG (H+L Antibody), Alexa Fluor 568 conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11011</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8242;, 6-Diamidin-2-phenylindol (DAPI)</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink PLA probe Anti-mouse PLUS</td>
			<td>Sigma</td>
			<td>DUO92001</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink PLA&#160; probe Anti-rabbit MINUS</td>
			<td>Sigma</td>
			<td>DUO92005</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink Detection Reagents Red</td>
			<td>Sigma</td>
			<td>DUO92008</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink Wash Buffers Fluorescence</td>
			<td>Sigma</td>
			<td>DUO82049</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink Mounting Medium with DAPI</td>
			<td>Sigma</td>
			<td>DUO82040</td>
			<td></td>
		</tr>
		<tr>
			<td>Mowiol 4-88</td>
			<td>Sigma</td>
			<td>81381</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>AX-70</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>SPOT</td>
			<td>Insight 2MP Firewire</td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray film</td>
			<td>Fujifilm</td>
			<td>Super RX</td>
			<td></td>
		</tr>
		<tr>
			<td>Film processing machine</td>
			<td>Fujifilm</td>
			<td>FPM-100A</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Figure 1 demonstrates that NF90 and RBM3 are both nuclear proteins and only a small fraction is present in the cytoplasm. Notably, there are three different bands stained positive for RBM3. The smallest just below 20 kDa reflects the correct size of RBM3 (the predicted molecular weight of RBM3 is 17 kDa). The origin of the two other bands remains to be investigated. Co-immunoprecipitation experiments with RBM3 as the bait protein revealed that NF90-RBM3 interactions are p...

Watch this Scientific Journal Video about Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay at JoVE.com

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.

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 ...

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