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

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

Pull-down assay for GST-eEF1A1 or eEF1A2(His)6 was carried out using GST-sepharose (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 times with 50 mM NaH2 PO4 , 300 mM NaCl, and 20 mM imidazole, to reduce nonspecific bound 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).
