Eif4a

Western blotting was performed according to standard methods. Cell lysates were extracted using NP-40 lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% NaDC, 0.1% SDS), and protein concentrations in the cell lysates were measured using the BCA Protein Assay kit (Thermo Scientific). Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. After incubation with PBS with 0.2% Tween 20 (PBST) containing 5% skimmed milk powder (SMP) for 1 h, the membranes were incubated overnight at 4 °C with primary antibodies diluted at 1:500–1:5000 in PBST containing 5% SMP. After washing five times with PBST, the membranes were incubated with secondary antibodies diluted at 1:10,000 in PBST containing 5% SMP at room temperature for 1 h. After washing five times again with PBST, the bound antibodies were visualized using ECL chemiluminescence (Thermo Scientific) on X-ray films (Carestream Health, Shanghai, China). Actin was used as a loading control. Antibodies against GABPB1 (1:1000), PRDX5 (1:1000), actin (1:5000), eIF4A (1:1000), Flag (1:2000) and horseradish peroxidase-conjugated secondary antibodies were purchased from ProteinTech (Chicago, IL, USA). Antibodies against caspase-3 (1:1000) and LC3 I/II (1:1000) were purchased from Cell Signaling Technology (Danvers, MA, USA).