HEK293T cells were cultured in 96-well plates in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in 5% CO2 and transfected with 200 ng of DNA by Lipofectamine LTX (Life Technologies) according to the manufacturer’s protocol. Antigen expression was detected as we described previously (43, 67). Briefly, at 48 hours after transfection, transfected cells were fixed with 4% formalin (Sigma), permeabilized with methanol at −20°C, and then blocked in 2.5% bovine serum albumin (Sigma) in phosphate-buffered saline (PBS) before the addition of anti-NS1 antibody (BioFront Technologies) at 37°C for 2 hours. Finally, cells were stained with DAPI (4′,6′-diamidino-2-phenylindole; Life Technologies) and visualized by fluorescence microscopy (Zeiss LSM-700), and the data were digitized using the Zen software (Zeiss).

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