FIG 8.

Lactosylceramide is associated with HCV NS4B protein. (A) Parental Huh7.5 and HCV Con1 (genotype 1b) replicon cells were grown for 48 h and processed for confocal microscopy with mouse monoclonal αLacCer antibody (1:500; green) and rabbit polyclonal αNS4B antibody (1:150; red). Huh7.5 cells also were infected with HCV Jc1 (MOI of 1) and processed for confocal microscopy as described above. The boxed areas represent a magnified view for colocalization (yellow) of lactosylceramide and HCV NS4B protein. (B) Huh7.5 cells were infected with HCV Jc1 (MOI of 1) as described for panel A and processed for confocal microscopy 24 h, 48 h, and 72 h postinfection. For each infection time point, confocal images were taken of 20 representative cells. The intensity of lactosylceramide pixels was calculated with the JACoP plugin in ImageJ software. Each filled circle or square (24 h; mock or Jc1 infected), upper or lower triangle (48 h; mock or Jc1 infected), diamond (72 h; mock), or open circle (72 h; Jc1 infected) represents one cell.