Enhanced chemiluminescent reagent

TA muscle samples were mechanically homogenized in RIPA buffer (0.1% SDS, 1% NP40, and 0.5% CHAPS) supplemented with protease inhibitors aprotinin, sodium orthovanadate, and phenylmethylsulfonyl fluoride (Sigma-Aldrich). Lysates were incubated on ice for 30 min and then centrifuged at 16.000 g, 4°C, for 20 min. The supernatant was collected, quantified via Bradford method (Bio-Rad), and stored in aliquots at −80°C to avoid repeated freezing-thawing cycles. For Western blot analysis, an equal amount of protein lysates (20–40 μg depending on the protein assayed) were separated onto Criterion™ TGX™ precast gels (Bio-Rad) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) using Criterion™ Blotter (Bio-Rad). Membranes were blocked with 5% BSA-TBS-T and then overnight incubated with primary antibodies at 4°C. Secondary antibody binding was performed at RT for 1 hour. After TBS-T washing, immunoreactive bands were incubated with enhanced chemiluminescent reagent (EuroClone) and detected via ChemiDoc™ XRS+ System (Bio-Rad). Densitometry analysis was accomplished through ImageJ software using H2B (nuclear), VDAC1 (mitochondrial), and β-actin (total) as protein normalizers. The results are representative of at least three independent experiments. The antibodies used are listed in Table 2.

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).