Anti eif4a

Total protein was extracted from tissues or cells using sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 25% glycerol, 5% β-mercaptoethanol, and 0.01% bromophenol blue) followed by heating at 100°C for 10 min. The protein extractions were separated by SDS-PAGE and transferred to the nitrocellulose filter (NC) membrane (Sangon Biotech, F619512). After being blocked with 5% non-fat milk in TBST for 1h at room temperature, membranes were incubated with desired primary antibodies diluting in 5% non-fat milk at 4°C overnight. Membranes were then washed three times with TBST and incubated with corresponding HRP-conjugated secondary antibodies for 1h at room temperature. The immunoblots were visualized after reaction with enhanced chemiluminescence (Vazyme, E412-02). Antibodies used in the experiments were presented as below: anti-β-Actin (Proteintech, 60008-1-Ig), anti-GAPDH (Proteintech, 60004-1-Ig), anti-METTL16 (Sigma, HPA020352), anti-Myc (CST, 2276), anti-Flag (Sigma, F1804), anti-eIF4E2 (Abclonal, A4305), anti-eIF4E (Santa Cruz, sc-9976), anti-eIF4A (Santa Cruz, sc-377315).