FIG. 4.

Immune activation by AcNPV is not mediated by gp64. (A) His-gp64ΔTM expressed in Sf-9 cells was purified and subjected to sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis under reducing conditions. Molecular size markers (lane M), purified AcNPV particles (lanes 1), and His-gp64ΔTM (lanes 2) were visualized by Coomassie blue (CBB) staining (left) and immunoblotting using antihexahistidine monoclonal antibody (middle) and anti-gp64 antibody (AcV5) (right). (B) PECs and splenic CD11c⁺ DCs (2 × 10⁵ cells/well) prepared from wild-type mice were stimulated with AcNPV (10 μg/ml) or His-gp64ΔTM (gp64) (20 μg/ml). After 24 h of incubation, production of IL-12 and IFN-α in culture supernatants was determined by ELISA. (C) MEFs (3 × 10⁵ cells/well) prepared from wild-type mice were stimulated with AcNPV (10 μg/ml) or His-gp64ΔTM (20 μg/ml). At 4 h or 8 h poststimulation, total RNA was extracted and expression of mRNA of IFN-β, IL-6, MCP-1, RANTES, and IP-10 was determined by real-time PCR. Data are shown as the means ± standard deviations.