FIG. 3.

Chemical inhibition of the clathrin pathway. Vero cells were treated with CPZ as specified in Materials and Methods. Cells were infected with BTV and harvested at 24 h p.i. (A) Immunoblotting of CPZ-treated cells was performed as previously described. To verify that the same amount of protein was loaded in each lane, the blots were also stained with an anti-β-tubulin antibody. The intensity of each band was quantified using ImageQuant software (GE Healthcare) and then normalized. (B) Inhibition of BTV infectivity in the presence of 5 and 10 μM of CPZ. Each sample was tested in triplicate. (C) The effect of CPZ was further confirmed by confocal microscopy. At 24 h p.i., cells were stained with a polyclonal anti-VP5 antibody and a FITC-conjugated secondary antibody (green). Nuclei were stained with Hoechst 33258 (blue). Images were collected as indicated in the legend to Fig. 1. Sample identification and final concentrations of the chemical are indicated.