**5.4. FRET analysis**

For Western blot analysis, cells (300 × 103

centrifuged at 14,000 × *g* for 20 min at 4°C.

Pull-down assay for GST-eEF1A1 or eEF1A2(His)6

PO4

**5.3. Confocal laser scanning microscopy**

using 60× oil immersion objective.

analyzed with mouse monoclonal anti-eEF1A antibody. For confocal microscopy and FRET analysis, cells (10 × 103

**5.2. Cytosolic extracts, pull-down assay, and Western blot**

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

74 Protein-Protein Interaction Assays

times with 50 mM NaH2

/well) were transfected with GST-eEF1A1 (1 μg),

) were layered on 10-mm glass cov-

was carried out using GST-sepharose

(1 μg) or with

(1 μg) and pcDNA3.1 (3 μg) as control using Lipofectamine 2000 or

, 300 mM NaCl, and 20 mM imidazole, to reduce nonspecific bound

K2. Twenty-four hours after transfection, cells were collected and the corresponding extract

After growth, HEK 293 were scraped, washed twice in PBS, resuspended for 30 min on ice in 20–40 μl of lysis buffer (50 mM Tris-HCl pH 7.4, 1% NP40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mg/ml aprotinin, leupeptin, pepstatin, 1 mM Na3VO4, 1 mM NaF), and then

(Amersham, Milan, Italy) or Ni-NTA agarose (Qiagen, Milan, Italy), respectively. In detail, 500 μg of cell extracts was incubated with pre-equilibrated resin (about 150 μl slurry/1 mg protein extract) for 2 h at room temperature (RT) or ON at 4°C, respectively. Subsequently, for GST pulldown, the resin was washed two times (centrifugation for 2 min at 2000 r.p.m. 4°C) with 1 ml of 1× phosphate-buffered saline (PBS), whereas for Ni-NTA pull-down, the resin was washed two

proteins, 0.05% Tween 20, pH 8.0. Successively, the samples were resuspended in 30 μl of 4× Laemmli loading buffer, heated to 95°C for 15 min and subjected to Western blot analysis.

Protein concentration was determined by a modified Bradford method, using the Bio-Rad protein assay and compared with bovine serum albumin (BSA) standard curve. Blots were developed using enhanced chemiluminescence detection (SuperSignal West Pico, Pierce, Milan, Italy). All films were scanned using Adobe Photoshop Software (San Jose, CA, USA).

Human embryonic kidney cells (HEK 293 Cell line) were treated for 20 min with glutaraldehyde 2.5% in PBS, washed three times with PBS, permeabilized for 10 min with 0.1% Triton-X100 and finally washed in PBS. Cells were then blocked for 20 min with 1% BSA in PBS, and after apposite washes, cells were incubated with rabbit anti-EF1A1 antibody (GenScript, Piscataway, NJ, USA) and mouse anti-His polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:300 in 1% BSA for 1 h. After washing three times with PBS, cells were incubated for 1 h with the appropriate secondary antibodies conjugated to fluorochromes and diluted 1:1000 in 1% BSA. Incubation with TOPRO 3-Iodide (Invitrogen Molecular Probes Eugene, OR, USA) diluted 1/1000 in BSA 1% was done for staining of the nucleus. After this, cells were washed properly with PBS and then observed with a Nikon Confocal Microscope C1 equipped with an EZ-C1 Software for data acquisition by

erslips, grown at confluence and then transfected with pcDNA3.1-eEF1A2(His)<sup>6</sup>

pcDNA3.1 (1 μg) as controls. Cells were analyzed after 24 h of incubation.

HEK 293 cells (7 × 103 cells/cm2 ) were grown for 24 h on glass coverslips under standard conditions (37°C, 5% CO<sup>2</sup> ). Cells were then rinsed with PBS, fixed for 10 min with formaldehyde (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 of the images.
