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. 2013 Jul 23;8(7):e69387. doi: 10.1371/journal.pone.0069387

Figure 5. Deletion of either of the three PRRSV nsp4 domains impaired its ability to induce apoptosis.

Figure 5

(A) Schematic diagram represents the PRRSV nsp4 protein deletion mutant constructs. (B) Expression of nsp4, N69, MID, and C157 proteins in Hela cells. Cells were transfected with pCMV-Myc, wild-type nsp4, or deletion mutant plasmids. At 48 hours post transfection, cells were lysed for western blotting to verify proteins expression using anti-myc antibodies. (C) Flow cytometry analysis of wild-type nsp4 and deletion mutants-induced apoptosis in Hela cells at 48 hours post transfection. (D) Percentage of apoptotic cells in panel C. (E) Double-labeling immunofluorescence analysis using TUNEL for apoptosis and indirect immunofluorescence for wild-type and mutant nsp4 proteins in Hela cells at 48 hours post transfection. Apoptosis (green), wild-type or mutant nsp4 (red), and nucleus (blue) were detected by immunofluorescence staining. (F) Percentage of apoptotic cells in panel E. Data represent means ± SD of three independent experiments. P<0.05 (*), P<0.01 (**), P<0.001 (***) as determined by student’s t test.