Sialic acids

The purified E protein was used in an enzyme-linked immunosorbent assay. The E protein (1 µg) was coated overnight at 4°C. The plates were blocked using 5% wt/vol BSA solution for 1 h at room temperature. The different concentrations of sialic acids (Solarbio) were added and incubated for 2 h. After washing, biotinylated SNA was added and incubated for 2 h. After washing again, HRP-conjugated streptavidin (Proteintech) was added to each well, and the plates were incubated for 1 h. The commercial peroxidase substrate system was used for signaling detection (SeraCare), and the optical density (OD) at 450 nm was measured with an ELISA reader.

S2 cells were transfected with recombinant plasmids and induced with 500 µM copper sulfate for 2 days. The cells were harvested and incubated with IP lysis buffer supplemented with protease inhibitor cocktail. The cell lysates were incubated with 2 µg sialic acids (Solarbio) at 4°C overnight, and subsequently, the mixture was incubated with 10 µg/mL biotinylated SNA. Dynabeads MyOne Streptavidin T1 (Invitrogen) was added for 2 h incubation. After washing with IP lysis buffer, the samples were detected by western blotting.