To prepare recombinant IRF7 (rIRF7) and p53 (rp53), the coding sequences of flounder IRF7 and p53 were amplified by PCR with primers F1/R1 and F2/R2, respectively (Table S1 ), and the PCR products were inserted into pET25951 (link) at the SwaI site. The proteins were expressed, purified, and cleared of endotoxin as reported previously51 (link). Mouse antibodies against rIRF7 and rp53 were prepared as reported previously51 (link) and purified using ProteinA/G Beads (Solarbio, Beijing, China).
IRF7 and p53 production in flounder spleen were examined by western blotting. Flounders were infected with megalocytivirus as described above for 4 days, after which the spleen protein were prepared as reported previously51 (link). Then, proteins were subjected to 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Watford, UK). The following steps were carried out according to previous study51 (link), of which the mouse antibodies against rPoIRF7 (1/4000 dilution) or rPop53 (1/2000 dilution) were used. Finally, protein bands were visualized using enhanced chemiluminescence Western Blotting Substrate (Promega, Madison, WI, USA).
IRF7 and p53 production in flounder spleen were examined by western blotting. Flounders were infected with megalocytivirus as described above for 4 days, after which the spleen protein were prepared as reported previously51 (link). Then, proteins were subjected to 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Watford, UK). The following steps were carried out according to previous study51 (link), of which the mouse antibodies against rPoIRF7 (1/4000 dilution) or rPop53 (1/2000 dilution) were used. Finally, protein bands were visualized using enhanced chemiluminescence Western Blotting Substrate (Promega, Madison, WI, USA).