4.3. Western Blotting Analysis

Cells were washed with phosphate-buffered saline (PBS) and incubated in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1:100 dilution of Sigma protease inhibitor mixture). Cell lysates were subjected to Western blot analysis after removing the insoluble fraction by centrifugation at 13,000× g for 20 min. The protein concentration of all extracts was determined using the Bradford assay. In total, 20 µg protein samples were subject to SDS-PAGE, and transferred to nitrocellulose membrane (Amersham Protran-GE Healthcare, Little Chalfont, Buckinghamshire, UK). After blocking nonspecific binding of antibody with 5% non-fat milk, blots were probed with one of the following antibodies: p53 (FL-393; Santa Cruz Biotechnology Inc. Dallas, Texas, USA), GAPDH (FL-335; Santa Cruz Biotechnology), β-Tubulin (D-10; Santa Cruz Biotechnology), Anti-Puromycin (12D10; Merck KGaA), eIF2α (D7D3; Cell Signaling Technology, Inc. Danvers, MA, USA), Phospho-eIF2α (Ser51) (D9G8; Cell Signaling), Anti-Hypusine Antibody (Merck Life Science), Anti-eIF5A (ab32014; Abcam), Anti-RACK1 (BD), Ribosomal Protein L30 (G-12; Santa Cruz Biotechnology), Ser-15 p53 were kindly provided by Prof Claudio Talora (University of Rome “La Sapienza”). Primary antibodies were detected by binding horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG-HRP (sc-2004; Santa Cruz Biotechnology), goat anti-mouse IgG-HRP (sc-2005; Santa Cruz Biotechnology) and using an enhanced chemiluminescent visualization system (ECL Western Blotting Substrate, Thermo Scientific-Pierce Biotechnology, Rockford, IL, USA). Primary and secondary antibodies were diluted according to the manufacturer instructions. The images were captured by a BioRad ChemiDoc™ MP Imaging system (Bio-Rad, Hercules, California, USA).