Figure 4. Arf6 is involved in baculovirus internalization and transgene expression.

(A) 293 cells were transfected with Arf6 constructs (wt, CA, DN) for 48 h and then transduced with baculovirus (AcVP39 MOI 200). The virus uptake after 30 min was detected by baculovirus Ab and Alexa-555-conjugated secondary antibody. The cell boundaries were determined from DIC image by ImageJ. The ratio of internalized vs. surface baculovirus in WT or Arf6DN transfected cells (30 cells from three separate experiments) was calculated from confocal sections using a differential labeling of baculovirus before and after permeabilization and using an internalization algorithm in BioimageXD software (see Materials and Methods section). Mean values and standard errors are shown. (B) Baculovirus-mediated luciferase (Luc; Ac-luc, MOI 200) expression in 293 cells was measured after 24 h in the presence Arf6 plasmids (left columns). Dextran internalization (2 h p.t.) in Arf6 expressing cells was measured to verify the efficacy of the mutant plasmids (right columns). The results show the proportional amount of cells which showed significant amount of dextran entry (cells with at least 10 dextran-positive vesicles in the cytoplasm). Mean values and standard deviations are calculated from three separate experiments. (C) Arf6 was knocked down in 293 cells using siRNAs. After siRNA treatments nuclear localization of baculovirus was calculated after 6 h baculovirus transduction. Western blotting was used to monitor the knock down effect with Arf6 antibodies. Statistical significance was determined by using the unpaired Student t test with a two-tailed P value. *P<0.05, **P<0.01, ***P<0.001.