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The glycoprotein of vesicular stomatitis virus promotes release of virus-like particles from tetherin-positive cells

Fig 2

VSV-G does not alter expression levels or cell surface localization of tetherin.

(A) Plasmids encoding tetherin and the indicated viral tetherin antagonists were transiently transfected into 293T cells. Transfection of empty plasmid (Mock) served as negative control. At 48 h post transfection, cells were harvested and stained for surface expression of tetherin using anti-tetherin antibody and Alexa-647 coupled secondary antibody. Staining was analyzed by flow cytometry. The graph shows relative surface expression values from three independent experiments, for which expression of tetherin on the surface of cells not expressing any antagonist (Mock) was set as 100%. Error bars indicate standard error of the mean (SEM). (B) 293T cells were transfected with plasmids encoding tetherin with an N-terminal c-Myc-tag and the indicated viral tetherin antagonists. At 48 h post transfection, tetherin expression in cell lysates was detected by Western blot analysis, using anti-c-Myc antibody. Detection of β-actin served as loading control. The results were confirmed in three separate experiments. Arrow heads indicate bands exhibiting the molecular weight expected for unglycosylated (white), partially (grey) and fully (black) N-glycosylated tetherin. (C) Quantification of four experiments performed as described for panel (B). The intensities measured for all tetherin signals with a molecular weight between 17 and 30 kDa were added to yield total tetherin expression. Tetherin expression in the absence of antagonist (mock) was set to 100%. Error bars indicate SEM. One-way ANOVA with Bonferroni post-test analyses were performed for panels A and C to test for statistically significant differences between samples with and without (Mock) antagonist (*, p ≤ 0.05).

Fig 2