Anti eif3a

Combined with a protease inhibitor cocktail and phosphatase inhibitor cocktail, RIPA lysis buffer was employed to make cells lysed. Protein concentrations were measured with a BCA kit. Separated by SDS‐PAGE, the protein was transferred onto the PVDF membranes. 5% BSA was used for blocking up to 2 h, the membranes then were incubated with primary antibodies at 4°C overnight. After the incubation and the subsequent washing for 30 min, membranes were incubated with HRP Goat Anti‐Rabbit IgG or HRP Goat Anti‐Mouse IgG antibodies at room temperature. Primary antibodies were as follows: anti‐YTHDF3 (ab220161, 1:1000 dilution, Abcam), anti‐LOXL3 (sc‐377216, 1:100 dilution, Santa‐Cruz), anti‐β‐actin (#4970, 1:1000 dilution, Cell Signaling Technology), anti‐eIF3A (ab128996, 1:1000 dilution, Abcam), anti‐METTL3 (15073‐1‐AP, 1:1000 dilution, Proteintech), anti‐METTL14 (26158‐1‐AP, 1:1000 dilution, Proteintech), anti‐FTO (27226‐1‐AP, 1:1000 dilution, Proteintech), anti‐ALKBH5 (16837‐1‐AP, 1:1000 dilution, Proteintech), anti‐CHD7 (A17180, 1:1000 dilution, ABclonal), anti‐PDE3A (A17919, 1:1000 dilution, ABclonal).

The anti-eIF4G, anti-ribosomal protein S5 (RPS5), anti-RPLP0, anti-eIF2a, anti-eIF3A, anti-eIF3e, anti-eIF4A, anti-eIF5B, and anti-RALY monoclonal antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-FLAG monoclonal antibody was purchased from Proteintech (Chicago, IL, USA). Anti-β-actin monoclonal antibody was purchased from Kangwei Century (Jiangsu, China). Anti-HA monoclonal antibody, the protein synthesis inhibitor cycloheximide (CHX), and secondary antibodies conjugated with horseradish peroxidase (HRP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Polyclonal pig antiserum directed against FMDV and polyclonal rabbit antiserum against SVA (Senecavirus A) and EMCV (Encephalomyocarditis virus) were generated and stored in our laboratory. Transfection reagents Lipofectamine LTX for transfection recombinant plasmids and Lipofectamine RNAiMax Reagent for small interfering RNA (siRNA) transfection were purchased from Invitrogen (CA, USA). The RNA extraction reagent TRIzol was purchased from Invitrogen.