**4. Conclusions**

within

that allowed discriminate proteins that colocalize in the same cellular compartment from those that are instead involved in specific molecular interactions. FRET effects were calculated using ImageJ plug-in software [24]. **Figure 4** shows the representation of the FRET effects where the blue color is indicated at low signal, whereas yellow-white color designated a high signal. The

**Figure 4.** FRET analysis of the interaction between eEF1A1 and eEF1A2. Representative pseudocolor images of cells labeled with rabbit anti-eEF1A1 and mouse anti-His primary antibodies followed by FITC and TRITC secondary-labeled

the cytoplasm with specific signals more intense especially at the level of the plasma membrane.

FRET is a powerful technique suitable for studying *in situ* interactions between biological molecules in cellular environments [25]. FRET can be assessed from the transfer of energy from one fluorescent molecule (donor) to another fluorescent molecule (acceptor). This process occurs optimally only if the two molecules are properly oriented and reasonably at a narrow distance (usually 1–10 nm) [26]. By this technique, the interaction between eEF1A1 and eEF1A2 in order to reinforce our hypothesis on the formation of an eEF1A1-eEF1A2 heterodimer [21] was highlighted. In a different approach, we afforded this aspect by expressing chimeric eEF1As fused to CFP and YFP as donor and acceptor (CFP-eEF1A1 and YFP-eEF1A2) in COS-7 cells, respectively [22]. However, some criticisms emerged that could have affected the FRET results such as (1) the possible interaction of the expressed chimeric proteins with endogenous enzymes, (2) self-association between eEF1A molecules (i.e., homodimer formation), and (3) the overexpression in COS-7 cells of both constructs that could have generated an art factual FRET signal mainly at the level of plasma membrane. Therefore, to overcome these concerns and to confirm that both eEF1A isoforms interact in the cellular environment, we used a different approach based on the use of IgG-FITC (donor)- and IgG-TRITC (acceptor)-conjugated antibodies. To this end, HEK 293 cell line, lacking the expression of eEF1A2 isoform, was transfected with

images clearly showed the interaction between eEF1A1 and transfected eEF1A2(His)6

**3. Discussion**

antibodies.

72 Protein-Protein Interaction Assays

Because eEF1A dimers are involved in actin bundling [31, 32], it emerges that the fraction of eEF1A as dimer is mostly involved in the actin cytoskeleton rearrangement. Therefore, the cellular distribution of eEF1A molecules between monomeric and dimeric form regulates the functional role of eEF1A in translation or in actin bundling. Because actin chains and translational system coexist in the cells and maybe also functionally dependent [33, 34], the transition "monomer-dimer-monomer" of eEF1A should be relatively easy depending on the cell conditions [35]. This interconversion may be regulated by the reversible posttranslational modifications of eEF1A [36] and its interactions with the protein partners such as Raf kinases [20, 21]. Therefore, it is possible that in cells coexpressing both isoforms, like cancer cells, eEF1A heterodimer formation could also be important for cytoskeleton rearrangements rather than for some phosphorylation catalysis most likely occurring during cell survival and apoptosis [20, 21].
