Figure 2
Effect of nitazoxanide on SeV protein synthesis. (A,B) Autoradiography of [35S]Met/Cys-labeled proteins (20 h-pulse; 4-24 h p.i., A) (1h-pulse; 23-24 h p.i., B) at 24 h p.i. from mock-infected or SeV-infected (3 PFU/cell) AGMK cells treated with 10 μg/ml NTZ, 10 μg/ml TIZ, 2.5 μg/ml tunicamycin (TM) or vehicle (C) after virus adsorption. Samples containing equal amount of radioactivity (A and B, left panel) or lysate (B, right panel) are shown. SeV proteins P (phosphoprotein), HN (hemagglutinin-neuraminidase), F0 (fusion glycoprotein F0 precursor), NP (nucleoprotein) and M (matrix) are indicated. Identification of SeV-F protein by immunoblot analysis (IB) in duplicate unlabeled mock-infected or SeV-infected samples is shown (B). (C) IB of SeV-F and α-tubulin levels at different times p.i. in samples treated as in (B). (D) IB for SeV-F, ubiquitin and β-actin in whole-cell extracts of mock-infected and SeV-infected AGMK cells treated for 24 h with NTZ (10 μg/ml), proteasome inhibitor bortezomib (BTZ) (25 nM) or vehicle. A moderate decrease in ubiquitinated protein levels is noted in bortezomib-treated cells in the presence of NTZ. (B–D) Full-length blots/gels are presented in Supplementary Figs 10 and 11.
