Fig. 4: SARS-CoV-2 Nsp1 inhibits translation in vivo.
From: SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation
a, Western blot analysis of HeLa cells expressing FLAG-Nsp1. HeLa cells were transfected with equal amounts of plasmid encoding either wild-type FLAG-tagged Nsp1 (WT) or FLAG-tagged Nsp1 with a mutated KH motif (KH). Cells transfected with empty vector served as the control (Ctrl). Whole-cell lysates were analyzed by western blotting using anti-FLAG and anti-GAPDH antibodies. b,c, Results of a puromycin incorporation assay in the presence of Nsp1. WT or KH FLAG-Nsp1 were transiently expressed in HeLa cells. Plasmid amounts were adjusted to achieve similar protein levels. As a control, one culture was incubated with cycloheximide (CHX). Before collection, nascent proteins were labeled with a puromycin pulse. Cell lysates were prepared and the Nsp1 expression (b, anti-FLAG) and incorporated puromycin (c, anti-puromycin) were analyzed by western blotting. GAPDH served as loading control. d, Quantification of puromycin incorporation. The intensity of each lane on the anti-puromycin stained blot (c) was measured and normalized to the intensity of the loading control. Bars represent mean values of three independent experiments (indicated by single dots) relative to the control transfection ± standard deviation (one-way ANOVA test P values: Ctrl versus WT, 0.0006; Ctrl versus KH, 0.9211; Ctrl versus CHX, <0.0001). Uncropped gel images for a–c and source data for d are available online.
