**5.1. Cell culture and transfection**

HEK 293 cells, obtained from the American Type Tissue Collection (Rockville, MD, USA), were grown at 37°C in a 5% CO<sup>2</sup> atmosphere in Dulbecco's modified Eagle medium (DMEM) (Gibco, Monza, Italy) supplemented with 10% heat-inactivated FBS (GIBCO), 100 U/ml penicillin, 100 mg/ml streptomycin, and 1% l-glutamine.

For Western blot analysis, cells (300 × 103 /well) were transfected with GST-eEF1A1 (1 μg), pcDNA3.1-eEF1A2(His)<sup>6</sup> (1 μg) and pcDNA3.1 (3 μg) as control using Lipofectamine 2000 or K2. Twenty-four hours after transfection, cells were collected and the corresponding extract analyzed with mouse monoclonal anti-eEF1A antibody.

**5.4. FRET analysis**

of the images.

**Acknowledgements**

**Conflicts of interest**

**Disclosure statement**

**Author contributions**

Nothing to declare.

manuscript.

HEK 293 cells (7 × 103

conditions (37°C, 5% CO<sup>2</sup>

cells/cm2

and POR Campania FSE 2007-2013, Project CRÈME.

The authors declare that there is no conflict of interest.

) were grown for 24 h on glass coverslips under standard

Cellular Interaction of Human Eukaryotic Elongation Factor 1A Isoforms

http://dx.doi.org/10.5772/intechopen.74733

75

). Cells were then rinsed with PBS, fixed for 10 min with form-

aldehyde (3.7% in PBS), permeabilized for 10 min with Triton X-100 (0.1% in PBS), and blocked for 20 min in bovine serum albumin (BSA) (1% in PBS). Subsequently, each sample was incubated for 1 h with 5 μg/ml of mouse anti-His and 5 μg/ml of human antieEF1A1 antibodies. Following PBS washes, cells were treated for 1 h with goat anti-mouse IgG FITC-conjugated antibody (donor) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1 μg/ml) and with goat anti-rabbit IgG-TRITC-conjugated antibody (acceptor) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (10 μg/ml), Finally, after 3× washes in PBS, confocal images were collected using a Nikon Confocal Microscope C1 furnished with EZ-C1 software. FRET analysis was carried out as already reported [24]. "FRET" images give the calculated amount of FRET for each pixel in the merged images. The ImageJ plug-in color codes the relative FRET efficiency, which is reported by the displayed color bar, on the right

This work was supported by funds from Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale (2012CK5RPF\_004), PON Ricerca e Competitività 2007-2013 (PON01\_02782),

NM, IR, and NMM were involved in WB analysis and cell extract preparation; GS and CS were involved in cell transfection; ER was in charge of tissue culture; VQ and FP performed confocal and FRET analysis; PA and AL were involved in the reading and approval of the

For confocal microscopy and FRET analysis, cells (10 × 103 ) were layered on 10-mm glass coverslips, grown at confluence and then transfected with pcDNA3.1-eEF1A2(His)<sup>6</sup> (1 μg) or with pcDNA3.1 (1 μg) as controls. Cells were analyzed after 24 h of incubation.
