Fig. 3

HSV-2 gD downregulates tetherin via lysosomal pathway. a HeLa cells transfected with pcDNA3.1 or plasmid expressing gD-flag/gM-flag were costained with anti-flag (red), anti-tetherin (purple) and anti-cathepsin D (green) antibodies. Nuclei were counterstained with Hoechst 33258 (blue). The colocalization of tetherin with lysosome markers (cathepsin D) was assessed by confocal microscopy. Representative confocal images from three independent experiments are shown. Scale bars in all panels represent 10 μm. b HeLa cells transfected with pcDNA3.1 or plasmid expressing gD-flag/gM-flag were costained with anti-flag (red), anti-tetherin (purple) and anti-20S proteasome (green) antibodies. Nuclei were counterstained with Hoechst 33258 (blue). The colocalization of tetherin with the proteasome markers (20S proteasome) was assessed by confocal microscopy. Representative confocal images from three independent experiments are shown. Scale bars in all panels represent 10 μm. c Pearson’s correlation coefficients were analyzed to determine the colocalization of tetherin and cathepsin D. d Pearson’s correlation coefficients were analyzed to determine the colocalization of tetherin and 20S proteasome. Data shown are mean ± SD by quantitative analyses of at least 20 distinct cells. e Western blot was used to analyze the expression of total tetherin in HeLa cells transfected with control plasmid and plasmid expressing gD-flag (treated or untreated with lysosome protease inhibitors (LPI)) where actin was used as a loading control. f HeLa cells transfected with pcDNA3.1 or plasmid expressing gD-flag were treated by lysis buffer. The prepared cell extract was ultracentrifuged by density gradient centrifugation and the lysosome band is located in the top 2 mL of the gradient. The corresponding bands were collected and the finally harvested lysosome pellets were detected by western blot. Samples 1 and 2 were two finally harvested lysosome pellets in two representative experiments