**2.3. Both eEF1A1 and eEF1A2 colocalize in HEK 293 cells**

**2.2. Both eEF1A1 and eEF1A2 immuno-interact after pull-down**

fected with pcDNA3.1 empty vector, cells transfected with eEF1A2(His)<sup>6</sup>

pcDNA3.1-eEF1A2(His)<sup>6</sup>

70 Protein-Protein Interaction Assays

cells with pcDNA3.1-eEF1A2(His)<sup>6</sup>

The possible interaction between eEF1A isoforms was analyzed by pull-down experiment. To this purpose, GST-eEF1A1 (kindly supplied by C. Sanges, Wurzburg, Germany [21]) and

transfection, cell extracts were analyzed by Western blot following GST-agarose and Ni-NTAagarose pull-down. As shown in **Figure 2**, compared to controls, GST pull-down of co-transfected cells showed the presence of a band of 54 kDa corresponding to the size of eEF1A2(His)<sup>6</sup> (**Figure 2A**, lane 2), whereas Ni-NTA pull-down showed the presence of a band of about 78 kDa corresponding to the size of the construct GST-eEF1A1 (**Figure 2B**, lane 2). **Figure 2B** (lane 3) also shows the presence of a band of about 26 kDa corresponding to the GST protein. This finding suggested that GST by itself somehow interacted with Ni-NTA matrix; thus, the result shown in line 2 could be partly due to an interaction of the GST moiety present in GST-eEF1A1 with Ni-NTA and not with eEF1A2. Therefore, to further confirm the interaction between eEF1A isoforms, a different approach was undertaken after transfection of HEK 293

**Figure 2.** Co-transfection of GST-eEF1A1 and eEF1A2-His in HEK 293 cells. GST-eEF1A1 and pcDNA3.1-eEF1A2(His)<sup>6</sup> were cotransfected in HEK 293 7 cells. After 24 h, the cells were harvested, lysed, and analyzed after GST pull-down with antibody anti-His (A) and after Ni-NTA pull-down with anti-GST antibody (B). (A) Lanes: 1, non-transfected

Ni-NTA pull-down with anti-eEF1A1, anti-eEF1A2, and anti-His antibody. (a–c) Lanes: 1, cells transfected with empty

; 3, cells transfected with GST and pcDNA3.1-eEF1A2(His)<sup>6</sup>

.

; 4, GST-agarose alone. (B) Lanes: 1, non-transfected cells; 2, cells transfected with GST-eEF1A1 and

was co-transfected in HEK 293 7 cells. After 24 h, the cells were harvested, lysed, and analyzed after

cells; 2, cells transfected with GST-eEF1A1 and pcDNA3.1-eEF1A2(His)<sup>6</sup>

vector; 2, cells transfected with pcDNA3.1-eEF1A2(His)<sup>6</sup>

eEF1A2(His)6

eEF1A2(His)6

pcDNA3.1-eEF1A2(His)<sup>6</sup>

constructs were co-transfected in HEK 293 cells and, after 24 h from

. In fact, as reported in **Figure 2C**, compared to cells trans-

showed, after Ni-NTA

; 3, cells transfected with GST and pcDNA3.1-

; 4, Ni-NTA alone. (C) pcDNA3.1-

The intracellular colocalization of eEF1A1 and eEF1A2 was first analyzed by confocal microscopy. As shown in **Figure 3**, HEK 293 cells after 48 h from transfection with pcDNA3.1 eEF1A2(His)6 construct revealed that both endogenous eEF1A (**Figure 3A**) and transfected eEF1A2(His)6 (**Figure 3B**) shared a cytoplasmic localization. The superimposition of the two panels (merged image, **Figure 3D**) showed that both eEF1A isoforms exhibited a cytoplasmic colocalization with specific signals more intense at the level of the plasma membrane.
