FIG. 4.

(A) Immunolocalization of VP1 (red) and Rab5 (green). 3T6 cells were infected with mouse polyomavirus (multiplicity of infection, 10² PFU per cell) and fixed 1 h postinfection. Immunofluorescence staining of VP1 (mouse polyclonal anti-VP1 antibody followed by Alexa Fluor-594 goat anti-mouse immunoglobulin) and a marker of early endosomes, Rab5 GTPase (rabbit polyclonal anti-Rab5 antibody and Alexa Fluor-488 goat anti-rabbit immunoglobulin) was performed. Merged confocal sections are shown. Bars, 5 μm. (B) Colocalization study of VP1 (red) and CTxB (green) or Tf (green) at 20°C. 3T6 cells were incubated with mouse polyomavirus (multiplicity of infection, 10² PFU per cell) and fluorescein isothiocyanate-labeled cholera toxin B-fragment (CTxB, 0.5 μg/ml) or transferrin-Alexa Fluor-488 (Tf, 50 μg/ml) at 20°C for 3 h. Cells were fixed, and VP1 was visualized by immunostaining (mouse polyclonal anti-VP1 antibody followed by Alexa Fluor-594 goat anti-mouse immunoglobulin). Merged confocal sections are shown. Bars, 5 μm.