**3. Discussion**

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 pcDNA3.1-eEF1A2(His)<sup>6</sup> , and the interaction of the recombinant eEF1A2(His)6 with endogenous eEF1A1 was assessed by pull-down, confocal microscopy and FRET analysis. The results obtained showed that the endogenous eEF1A1 and the expressed eEF1A2 interacted in HEK 293 cells at the level of both cytoplasm and plasma membrane. Moreover, the FRET image highlighted a more intense signal at the level of the plasma membrane. These data confirmed those reported in our previous work [22], thus strongly confirming the association in the cells of eEF1A isoforms.

The homodimer association of eEF1As has recently emerged from the crystallization of rabbit eEF1A2 [27] or as proposed in *Tetrahymena*, in order to explain actin bundling essential for the regulation of actin cytoskeleton and cell morphology during several cellular processes [16]. The possible association between eEF1A isoforms was instead proposed by Sanges et al. [21] in studying the control of eEF1A function in cancer cells *via* phosphorylation and by Lee et al. [28] in studying the interaction of eEF1A2 with the tumor suppressor protein p16INK4a. Since eEF1A1 and eEF1A2 display a very high amino acid sequence identity (above 97%), the overall structures appear quite similar, as can be predicted by bioinformatic analysis at the GRAMM-X docking Web Server v.1.2.0 [29, 30], using rabbit eEF1A2 (PDB 4C0S chain A) as template [27]. These considerations suggest that both eEF1A1 and eEF1A2 complexes are present in the cells either as homodimer or as heterodimer. These complexes are most likely associated with regulatory noncanonical functions of eEF1As.
