FIG. 2.

Characterization of BSC1 cells stably expressing the VP3 M3 deletion mutant polypeptide. BSC1 cells expressing the M3 polypeptide were generated by transfection with plasmid pCI-neo/M3 and subsequent selection in the presence of G418. (A) Immunofluorescence analysis of three selected clones, BSC1/M3L (L), BSC1/M3M (M), and BSC1/M3H (H). Cells were grown on coverslips, fixed, and incubated with rabbit anti-VP3 serum, followed by incubation with goat anti-rabbit immunoglobulin coupled to Alexa 594 (red). Cell nuclei were stained with ToPro-3 (blue). Samples were visualized by epifluorescence using a Zeiss Axiovert 200 microscope equipped with a Bio-Rad Radiance 2100 confocal system. Images were captured using the Laser Sharp software package (Bio-Rad). (B) Cultures of BSC1/M3L (L), BSC1/M3M (M), and BSC1/M3H (H) were harvested and analyzed by Western blotting using a mixture of mouse anti-human β-actin and rabbit anti-VP3 sera, followed by addition of a mixture of horseradish peroxidase-conjugated goat anti-mouse and rabbit anti-rabbit immunoglobulins. Signals were detected by incubation with 1-chloro-4-naphthol in the presence of hydrogen peroxide. The positions of the β-actin and M3 polypeptides are indicated.