Anti flag monoclonal antibody

The ssDNA oligonucleotides and SARS-CoV-2 nucleocapsid protein coding sequences used in this study were synthesized by Beijing Aoke Dingsheng Biotechnology Co., Ltd. The specific sequences of the ssDNA oligonucleotides are provided in Table S1. The following reagents and kits were purchased from the respective suppliers: isopropyl β-D-thiogalactoside (IPTG), kanamycin, Lipo293 Transfection Reagent, Bradford Protein Concentration Determination Kit, the His-tag Purification Resin, Alexa Fluor 555-labeled Donkey Anti-Rabbit (H + L) antibody, Anti-Flag Magnetic Beads, and Anti-His Magnetic Beads were obtained from Beyotime Co., Ltd. Taq DNA polymerase, T4 DNA ligase, and CCK-8 Cell Counting Kit were purchased from Vazyme Biotechnology Co., Ltd. Anti-Flag Monoclonal Antibody, Anti-His Monoclonal Antibody, and anti-GAPDH Monoclonal Antibody were purchased from Proteintech Group, Inc. Dulbecco’s modified Eagle’s medium (DMEM; high glucose) and penicillin-streptomycin (liquid) were obtained from Thermo Fisher Scientific, Inc.

Equal numbers of OV cells or NC cells were transfected with equal amount of pFLAG‐CMV™‐2‐MKK4 and pUb‐HA (a plasmid for overexpressing Ub with HA‐tag from Dr Chengjiang Gao's Lab, Shandong University School of Basic Medical Sciences). After 48 hourrs post‐transfection, the cells were harvested for immunoprecipitation as routinely used. The antibody used to pull down protein was anti‐flag monoclonal antibody from ProteinTech Group. The beads with protein A and protein G were purchased from Solarbio Science & Technology. The pull‐down samples were applied for Western blotting with 12CA5 (an anti‐HA monoclonal antibody).

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.