Pcbp2

Human embryonic kidney (HEK293T) cells and the porcine kidney cell line (PK-15) (ATCC) were cultivated in Dulbecco’s modified eagle’s medium supplemented with 10% fetal bovine serum, 100 U penicillin/ml, and 100 μg streptomycin/ml in a humidified incubator with 5% CO₂ at 37 °C. Visa^–/– and wild-type mouse embryonic fibroblasts (MEFs) were kindly provided by Dr. Hong-bing Shu (Wuhan University). The antibodies used in this study were as follows: rabbit ployclonal antibodies against PCBP2 (Abcam), IgG (Ig) (Sigma), and VISA (CST); mouse monoclonal antibodies against flag, Myc, IgG (Ig), β-actin, GST (Sigma), HA (OriGene), and GFP (Thermo Fisher Scientific). Mouse anti-VP3 sera were prepared in our laboratory using a recombinant FMDV VP3 protein. Sendai virus (SeV) inducing the activation of interferon was previously described^{18 (link)}. The type O FMDV was propagated in PK-15 cells, and the supernatants of the infected cells were clarified and stored at –80 °C.

Cells were lysed for 1 h at 4 °C with 1% (vol/vol) Nonidet P-40 in PBS with a protease-inhibitor 'cocktail', separated by SDS-PAGE and transferred onto polyvinylidenedifluoride (PVDF) membranes. The membranes were blocked for 1 h at room temperature with 5% non-fat milk in Tris-buffered saline (TBS) plus Tween 20 (TBST), followed by an overnight incubation at 4 °C with an antibody (diluted in blocking buffer) against PCBP2 (Abcam). Normalization was performed by blotting the same samples with an antibody against GAPDH.