Name: Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells (Video) | JoVE Journal
Uploaded: 2020-05-01T13:30:00.000+00:00
Duration: 8 min 47 s
Description: Learn to measure eIF4E-eIF4G protein-protein interactions in live HEK 293 cells using a cell-based complementation assay. This method enables assessment of drug-induced perturbations in eIF4F complex dynamics for cancer research.

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5 minutes

This work was supported by core budget from the p53lab (BMSI, A*STAR) and the JCO VIP grant (A*STAR).

HEK293 cell line was used for the protocol and was cultured in Dulbecco&#39;s Modified Eagle Medium supplemented with 10% Fetal Bovine Serum, 2 mM L-glutamine, and 100 U/mL penicillin/streptomycin. Cells were cultured at 37 &#176;C with 5% CO2 in a humidified environment.
1. Quantitative assessment of eIF4F complex disruption via eIF4E:eIF4G604-646 complementation assay
<ol>
	<li>Cell culture and transient transfection of eIF4E:eIF4G604-646 complementation assay
	<ol>
		<li>Use freshly thawed cells with less than 20 passages for all experiments. On day 1, determine the cell number using a standard cell counter and count the total viable cells using the Trypan blue exclusion method13.</li>
		<li>Seed 6-well plates with 0.9 - 1.2 x 106 of HEK 293 cells per well in 2 mL of standard growth medium. 
		NOTE: In order to achieve the best transfection efficiency within the cell lines indicated above, ensure that the plated cells are 70-90% confluent the day after seeding.</li>
		<li>On the morning of day 2, co-transfect cells with SubA-eIF4E and eIF4G604-646-SubB plasmid using a lipid-based transfection reagent (see Table of Materials) as described below.
		<ol>
			<li>Dilute 9 &#181;L of liposome-based solution in a tube containing 125 &#181;L of reduced serum medium without phenol red (see Table of Materials) for each transfection and incubate at room temperature for 5 min.</li>
			<li>Prepare master mix of DNA by diluting 3 &#181;g of each plasmid in 125 &#181;L of reduced serum medium for each transfection tube.</li>
			<li>Add 12 &#181;L of enhancer reagents to the DNA master mix tube, mix well and immediately add the DNA: enhancer master mix to each tube of diluted liposomes in a ratio 1:1. Incubate for 15 min at room temperature.</li>
			<li>Add the DNA-lipid complex to each well and incubate the cells in a 37 &#176;C incubator with 5% CO2 for 24 h.</li>
		</ol>
		</li>
		<li>On the morning of day 3, rinse each well with 1 mL of PBS.</li>
		<li>Remove the PBS and incubate cells in each well with 0.3 mL of trypsin for 5 min at 37 &#176;C.</li>
		<li>Neutralize trypsin by adding 2 mL of the reduced serum medium without phenol red containing 0% FCS in each well and transfer transfected cells into a 15 mL tube. 
		NOTE: Phenol red can interfere with the luciferase activity; therefore, cells must be handled from this step onward in medium without phenol red.</li>
		<li>Spin down cells for 5 min at 290 x g and aspirate the medium. Resuspend the cells in 2 mL of the reduced serum medium without phenol red containing 0% FCS. Count as described in step 1.1.</li>
		<li>Seed transfected HEK 293 cells in 96 well opaque plates at a density of 30,000 cells per well in 90 &#181;L of medium without phenol red containing 0% FCS. In order to obtain 3 technical replicates within the same experiment for 3 different compounds, seed 60 wells of the plates with cells excluding the wells on the edges.</li>
		<li>Immediately after seeding the transfected cells, add 10 &#181;L of 10% DMSO compound solution (see step 1.2).</li>
	</ol>
	</li>
	<li>Compound preparation
	<ol>
		<li>Prepare 1 mM compound stock solutions by dissolving the compound of interest in 100% DMSO (v/v). In order to have 3 replicates for each compound titration, use 8 &#181;L of the 1 mM compound stock solution. 
		NOTE: The volume of 1 mM stock compound used is more than what it is needed. This is done to take into account pipetting errors if any.</li>
		<li>Perform a 2-fold serial dilution of the stock compound solution with 4 &#181;L of the 1 mM stock into 4 &#181;L of 100% DMSO for each titration point. 
		NOTE: For a complete compound titration, perform 9 serial dilutions from the starting stock in 100% DMSO. If multiple compounds need to be tested, use a 96 well plate and a multichannel pipette to facilitate this step and subsequent dilutions.</li>
		<li>Add 36 &#181;L of HPLC grade sterile water for each point of the 2-fold serial dilution to prepare a 40 &#181;L of 10x working solutions in 10% DMSO (v/v) (i.e. 100 &#956;M to 0.39 &#956;M 10% DMSO stock series will lead to a final solution of 10 &#956;M to 0.039 &#956;M,in 1% DMSO when added to cells). As for treatment control, also prepare a 10% DMSO only stock solution in HPLC grade sterile water.</li>
		<li>Add 10 &#181;L of 10x working solutions to the cells in the 96 well opaque plate in order to yield the intended final concentration with a residual DMSO concentration of 1% (v/v) in a total volume of 100 &#181;L and incubate for 3 h at 37 &#176;C with 5% v/v atmospheric CO2.</li>
	</ol>
	</li>
	<li>Luciferase complementation and viability assay
	<ol>
		<li>After 3 h of drugging, start preparing the luciferase substrate reagent by combining 1 volume of substrate with 19 volumes of the dilution reagent (see manufacturer&#39;s instructions). 
		NOTE: The luciferase assay is performed using a commercially available kit (see Table of Materials).</li>
		<li>Use a multichannel pipette to immediately add 25 &#181;L of substrate reagent.</li>
		<li>Shake the plate at 350 rpm for 50 min on an orbital shaker at room temperature.</li>
		<li>Assess luminescence using a plate reader. To do so, set the mirror reader on luminescence and the emission filter on 455. Use a measurement height of 6.5 mm with a measurement time of 1 s.</li>
		<li>In order to asses cell viability, add 33 &#181;L of viability assay reagent and then re-measure luminescence again after 15 min at room temperature, using the plate reader.</li>
		<li>Assess luminescence with the plate reader by setting the mirror reader on Luminescence and the emission filter on 600 nm. Use a measurement height of 6.5 mm with a measurement time of 1 s. 
		NOTE: Viability is assessed by measuring the intracellular level of ATP upon cell lysis as per manufacturer instructions. Multiplexing is possible in this case since the viability assay uses a different luciferase with a different emission wavelength.</li>
		<li>Use the data to determine the IC50 values of each compound by curve fitting the data to a 4 parameter fitting curve equation: 
		Y = ((A-D)/(1+((x/C)^B))) + D 
		The numerical values D and A must be constrained in the curve fitting process: 
		D = luminescent value on the Y-axis for minimal curve asymptote or minimal theoretical level of response expected from the eIF4E:4G complementation assay. 
		A = luminescent value on the Y-axis for maximal curve asymptote or maximal theoretical level response expected from the eIF4E:4G complementation assay. 
		NOTE: The value for the minimal response is derived from the 1% DMSO (v/v) control treatment and the value for the maximal response is derived from the maximal response of a high affinity mTOR inhibitor that has plateaued with no effect on cell viability.</li>
	</ol>
	</li>
</ol>
2. Correlating eIF4E:eIF4G604-646 assay inhibition with eIF4F complex disruption in cells
<ol>
	<li>To confirm that the signal being measured by the assay corresponds to the physical disruption of eIF4E-eIF4G interaction by the compound, seed cells as described in step 1.1.</li>
	<li>The day after, replace the medium with 1 mL of reduced serum medium not containing phenol red and incubate for 4 h with the compound of interest. Ensure residual DMSO concentration of 1% (v/v) in a total volume of 1 mL. 
	NOTE: In order to easily allow a correlation with the result, use compound concentrations at the beginning, midpoint and endpoint of the measured complementation assay titration curve.</li>
	<li>Lyse cells and performed m7GTP pull down to isolate eIF4F and eIF4E:4EBP1 complexes. Detailed procedures on how to perform the m7GTP pull down experiment can be found elsewhere14.</li>
	<li>Detect m7GTP bound eIF4G, eIF4E and 4EBP1 protein levels by western blot analysis, and then correlate to eIF4F complex disruption with complementation assay signal inhibition.</li>
</ol>

<ol><li>Silvera, D., Formenti, S. C., Schneider, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Translational+control+in+cancer%5BTitle%5D)+AND+%22Nature+Reviews+Cancer%22%5BJournal%5D)">Translational control in cancer.</a> Nature Reviews Cancer. 10 (4), 254-266 (2010).</li>
<li>Gebauer, F., Hentze, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+Mechanism+of+translational+control%5BTitle%5D)+AND+%22Nature+Reviews+Molecular+Cell+Biology%22%5BJournal%5D)">Molecular Mechanism of translational control.</a> Nature Reviews Molecular Cell Biology. 10, 827-835 (2004).</li>
<li>Bhat, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+the+translation+machinery+in+cancer%5BTitle%5D)+AND+%22Nature+Reviews+Drug+Discovery%22%5BJournal%5D)">Targeting the translation machinery in cancer.</a> Nature Reviews Drug Discovery. 14 (4), 261-278 (2015).</li>
<li>Pelletier, J., Graff, J., Ruggero, D., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+the+eIF4F+translation+initiation+complex%3A+a+critical+nexus+for+cancer+development%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Targeting the eIF4F translation initiation complex: a critical nexus for cancer development.</a> Cancer Research. 75 (2), 250-263 (2015).</li>
<li>Montanaro, L., Pandolfi, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Initiation+of+mRNA+translation+in+oncogenesis%3A+the+role+of+eIF4E%5BTitle%5D)+AND+%22Cell+Cycle%22%5BJournal%5D)">Initiation of mRNA translation in oncogenesis: the role of eIF4E.</a> Cell Cycle. 11, 1387-1389 (2004).</li>
<li>Merrick, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((eIF4E%3A+A+retrospective%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">eIF4E: A retrospective.</a> Journal of Biological Chemistry. 290 (40), 24091-24099 (2015).</li>
<li>Marcotrigiano, J., Gingras, A. C., Sonenberg, N., Burley, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cap-dependent+translation+initiation+in+eukaryotes+is+regulated+by+a+molecular+mimic+of+eIF4G%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">Cap-dependent translation initiation in eukaryotes is regulated by a molecular mimic of eIF4G.</a> Molecular Cell. 3 (6), 707-716 (1999).</li>
<li>Umenaga, Y., Paku, K. S., In, Y., Ishida, T., Tomoo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+and+function+of+the+second+eIF4E-binding+region+in+N-terminal+domain+of+eIF4G%3A+comparison+with+eIF4E-binding+protein%5BTitle%5D)+AND+%22Biochemical+and+Biophysical+Research+Communication%22%5BJournal%5D)">Identification and function of the second eIF4E-binding region in N-terminal domain of eIF4G: comparison with eIF4E-binding protein.</a> Biochemical and Biophysical Research Communication. 414 (3), 462-467 (2011).</li>
<li>Grüner, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Structures+of+eIF4E-eIF4G+Complexes+Reveal+an+Extended+Interface+to+Regulate+Translation+Initiation%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">The Structures of eIF4E-eIF4G Complexes Reveal an Extended Interface to Regulate Translation Initiation.</a> Molecular Cell. 64 (3), 467-479 (2016).</li>
<li>Gingras, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hierarchical+phosphorylation+of+the+translation+inhibitor+4E-BP1%5BTitle%5D)+AND+%22Genes+and+Development%22%5BJournal%5D)">Hierarchical phosphorylation of the translation inhibitor 4E-BP1.</a> Genes and Development. 15 (21), 2852-2864 (2001).</li>
<li>Dixon, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NanoLuc+Complementation+Reporter+Optimized+for+Accurate+Measurement+of+Protein+Interactions+in+Cells%5BTitle%5D)+AND+%22ACS+Chemical+Biology%22%5BJournal%5D)">NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells.</a> ACS Chemical Biology. 11 (2), 400-408 (2016).</li>
<li>Sun, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((mTOR+kinase+inhibitors+as+potential+cancer+therapeutic+drugs%5BTitle%5D)+AND+%22Cancer+Letters%22%5BJournal%5D)">mTOR kinase inhibitors as potential cancer therapeutic drugs.</a> Cancer Letters. 340 (1), 1-8 (2013).</li>
<li>Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trypan+blue+exclusion+test+of+cell+viability%5BTitle%5D)+AND+%22Current+Protocols+in+Immunology%22%5BJournal%5D)">Trypan blue exclusion test of cell viability.</a> Current Protocols in Immunology. , Appendix 3: Appendix 3B (2001).</li>
<li>Sekiyama, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+mechanism+of+the+dual+activity+of+4EGI-1%3A+Dissociating+eIF4G+from+eIF4E+but+stabilizing+the+binding+of+unphosphorylated+4E-BP1%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Science+U.+S.+A%22%5BJournal%5D)">Molecular mechanism of the dual activity of 4EGI-1: Dissociating eIF4G from eIF4E but stabilizing the binding of unphosphorylated 4E-BP1.</a> Proceedings of the National Academy of Science U. S. A. 112 (30), e4036-e4045 (2015).</li>
<li>Muller, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((4E-BP+restrains+eIF4E+phosphorylation%5BTitle%5D)+AND+%22Translation%22%5BJournal%5D)">4E-BP restrains eIF4E phosphorylation.</a> Translation. 1 (2), e25819(2013).</li>
<li>Frosi, Y., Usher, R., Lian, D. T. G., Lane, D. P., Brown, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monitoring+flux+in+signalling+pathways+through+measurements+of+4EBP1-mediated+eIF4F+complex+assembly%5BTitle%5D)+AND+%22BMC+Biology%22%5BJournal%5D)">Monitoring flux in signalling pathways through measurements of 4EBP1-mediated eIF4F complex assembly.</a> BMC Biology. (1), 40(2019).</li>
<li>Ran, X., Gestwicki, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibitors+of+protein-protein+interactions+%28PPIs%29%3A+An+analysis+of+scaffold+choices+and+buried+surface+area%5BTitle%5D)+AND+%22Current+Opinion+in+Chemical+Biology%22%5BJournal%5D)">Inhibitors of protein-protein interactions (PPIs): An analysis of scaffold choices and buried surface area.</a> Current Opinion in Chemical Biology. 44, 75-86 (2018).</li>
</ol>

The authors have nothing to disclose.

The method described in this article utilizes a luciferase-based complementation assay to quantitatively monitor eIF4F complex assembly through direct measurement of eIF4G-eIF4E interaction in live cells. We have provided details for use of eIF4E-eIF4G complementation system and we have also showed that the system is extremely accurate in measuring drug-induced 4EBP1-mediated dissociation of eIF4E-eIF4G interaction16. In order to facilitate the throughput of this&#160;assay, the experimental setup...

Control of gene expression plays a pivotal role in the correct execution of cellular programs such as growth proliferation and differentiation. A regulatory control mechanism can be exerted either at the level of gene transcription or at the level of mRNA translation. In the last decade, it has become increasingly evident that translational control by modulation of the initiation process rather than the later steps of elongation and termination can finely regulate synthesis of specific subsets of proteins that play a wide range of biological functions.
Increased translation of mRNAs involved in survival, anti-autophagic and anti-apoptotic responses have been implicated in several cancers and have also been causatively linked to either aberrant activation or over expression of translation initiation factors1.
The eIF4F complex is a master regulator of translation initiation. By binding the cap-structure on the 5&#39; end of mRNAs, eIF4F is driving initial mRNA-ribosome recruitment and in turn increasing mRNA translation efficiency of weakly translated eukaryotic mRNAs2. eIF4F mediated translation of cancer-related mRNAs has been reported for many cancer models harboring aberrant activation of RAS/MAPK or AKT/TOR pathways, suggesting that cancer cells upregulate eIF4F to boost their own pro-neoplastic activity. Disruption of this feed-forward loop by inhibiting eIF4F complex formation is thereby a very promising therapeutic strategy3,4.
The eIF4F complex consists of (i) eIF4E, the cap-binding subunit of eIF4F that interacts with the cap structure found at the 5&#39; UTR of mRNA, (ii) eIF4A, the RNA helicase and (iii) eIF4G, the scaffold protein that interacts with both eIF4A and eIF4E and which eventually recruits the 40S ribosomal subunit5. eIF4G association with eIF4E is the rate-limiting step for the assembly of functional eIF4F complexes and it is negatively regulated by the eIF4E binding proteins (4EBPs, members 1, 2 and 3))6. By competing with eIF4G binding to eIF4E through an interface that consists of canonical and non-canonical eIF4E binding sequences7,8,9 (region spanning aa 604-646 on human eIF4E), 4EBP reduces the pool of eIF4E actively involved in translation and preventing eIF4F complex formation. Interplay of these protein-protein interactions is mainly regulated by the mammalian target of rapamycin (mTOR)-mediated phosphorylation of 4EBP. Upon mitogenic stimuli, mTOR directly phosphorylates the members of the 4E-BP protein family, decreasing their association with eIF4E and, thereby, promoting eIF4E-eIF4G interaction and formation of functional eIF4F complexes10.
Despite the great effort in developing compounds targeting eIF4F complex integrity, the lack of assays measuring direct disruption of eIF4E-eIF4G interaction in live cells has limited the search for cellular active hit compounds. We have applied a luciferase assay based on a coelenterazine analog (e.g., Nanoluc-based complementation assay) to monitor in real time the status of eIF4F integrity through the eIF4E-eIF4G interaction. The luciferase complementation protein system consists of an 18 kDa protein fragment (SubA) and 11 amino acid peptide fragment (SubB) optimized for minimal self-association and stability11.&#160;Once expressed as a fusion product with the human full length eIF4E and the eIF4E interaction domain from human eiF4G1 (aa 604-646), the two interacting proteins will bring the SubA and SubB fragment into close proximity of each other and will induce the formation of the active luciferase that, in presence of a cell permeable substrate, will eventually generate a bright luminescent signal (Figure 1). We have reported elsewhere the construction and validation of the eIF4E:eIF4G604-646 complementation system16.
Here, we describe how the eIF4E:eIF4G604-646 complementation system (available upon request) can be applied to accurately measure 4EBP1-mediated eIF4E-eIF4G disruption in live cells. Additionally, we demonstrate its utility by measuring the effects of several mTOR inhibitors that are currently under clinical trials as potential cancer therapeutic drugs12. Because off-target&#160;effects often mask drug-specific activity, we also describe how the versatility of the eIF4E:eIF4G604-646 system measurement can be extended with orthogonal measurements of cellular viability to take these into account.

<table><tbody><tr><td>293FT cells</td><td>Thermo Fisher Scientific</td><td>R70007</td><td></td></tr><tr><td>Cell culture microplate 96 well, F-Bottom</td><td>greiner bio-one</td><td>655083</td><td></td></tr><tr><td>Cell titer Glo 2.0</td><td>PROMEGA</td><td>G9241</td><td></td></tr><tr><td>Envision Multilabel Reader</td><td>PerkinElmer</td><td>not applcable</td><td></td></tr><tr><td>Finnpipette F2 Multichannel Pipettes 12-channels 30-300 ml</td><td>Thermo Fisher Scientific</td><td>4662070</td><td></td></tr><tr><td>Finnpipette F2 Multichannel Pipettes 12-channels 5-50 ml</td><td>Thermo Fisher Scientific</td><td>4662050</td><td></td></tr><tr><td>FUGENE6</td><td>PROMEGA</td><td>E2692</td><td></td></tr><tr><td>Lipofectamine 3000</td><td>Thermo Fisher Scientific</td><td>&lt;a href=&quot;https://www.thermofisher.com/order/catalog/product/L3000015&quot; target=&quot;_blank&quot;&gt;L3000015&lt;/a&gt;</td><td></td></tr><tr><td>NanoBiT PPI Starter Systems</td><td>PROMEGA</td><td>N2014</td><td></td></tr><tr><td>Optimem I Reduced Serum Mediun, no phenol red</td><td>Thermo Fisher Scientific</td><td>11058021</td><td></td></tr><tr><td>Orbital shaker</td><td>Eppendorf</td><td>not appicable</td><td></td></tr><tr><td>&amp;gamma;-Aminophenyl-m&lt;sup&gt;7&lt;/sup&gt;GTP (C&lt;sub&gt;10&lt;/sub&gt;-spacer)-Agarose</td><td>Jena Bioscience</td><td>AC-155S</td><td></td></tr></tbody></table>

monitoring eif4f assembly by measuring eif4e-eif4g interaction in live cells

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo&#160;oncogenic activation in humans. eIF4F is a cap-binding complex involved in the mRNA-ribosome recruitment phase of translation initiation. In many cellular and pre-clinical model of cancers, the deregulation of eIF4F leads to increased translation of specific mRNA subsets that are involved in cancer proliferation and survival. eIF4F is a hetero-trimeric complex built from the cap-binding subunit eIF4E, the helicase eIF4A and the scaffolding subunit eIF4G. Critical for the assembly of active eIF4F complexes is the protein-protein interaction between eIF4E and eIF4G proteins. In this article, we describe a protocol to measure eIF4F assembly that monitors the status of eIF4E-eIF4G interaction in live cells. The eIF4e:4G cell-based protein-protein interaction assay also allows drug induced changes in eIF4F complex integrity to be accurately and reliably assessed. We envision that this method can be applied for verifying the activity of commercially available compounds or for further screening of novel compounds or modalities that efficiently disrupt formation of eIF4F complex.

In order to validate the sensitivity of the eIF4E:eIF4G604-646 complementation system, 4EBP1 mediated inhibition of eIF4F complex assembly was assessed by using mTOR inhibitors. By inhibiting mTORC1 kinase dependent phosphorylation of the 4EBP protein family, mTOR inhibition enhances 4EBP1 association to eIF4E and, therefore, eIF4F disassembly15. Two classes of mechanistically different inhibitor of mTOR kinases were tested: rapalogs (e.g., Rapamycin) th...

Here, we present a protocol to measure eIF4E-eIF4G interaction in live cells that would enable the user to evaluate drug induced perturbation of eIF4F complex dynamics in screening formats.

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo&#160;oncogenic ...

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Learn to measure eIF4E-eIF4G protein-protein interactions in live HEK 293 cells using a cell-based complementation assay. This method enables assessment of drug-induced perturbations in eIF4F complex dynamics for cancer research.

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

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