Western Blot Analysis of aIF5A and Hypusine

Western blot analysis was performed as previously described (Bassani et al., 2018 (link)) with some modifications. Briefly, 15 μg of total proteins for each sample was separated on SDS-15% polyacrylamide gel using standard protocols and then transferred onto a 0.2-μm nitrocellulose membrane (GE Healthcare) using wet transfer blotting apparatus. Protein transfer was performed at 100 V for 30 min in a transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol). Nonspecific binding was blocked using 5% nonfat milk. The membranes were probed overnight at 4°C, either with anti-aIF5A (used at a 1:5,000 dilution in TBS-Tween containing 5% of nonfat milk) or with anti-hypusine antibody (Millipore). The detection of primary antibodies was obtained by horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Cell Signaling Technology), using the enhanced chemiluminescent reagent (EuroClone). The images were visualized with a BioRad ChemiDoc™ MP Imaging System. The quantification of the signals was obtained by the ImageLab™ software (Biorad).