Fig 4.

Expression of viral ORF4 protein in wPCV2-infected PK-15 cells and mice. At 60 hpi with wPCV2, PK-15 cells (arrow) were fixed and probed with an anti-Cap MAb (A) and anti-ORF4 2D3 MAb (B) for confocal microscopic analysis. Nuclei were labeled with DAPI (bars, 25 μm). At 21 dpi, the 2D3 MAb to the PCV2 ORF4 protein was used to stain (green) cryosectioned spleens of wPCV2-inoculated mice (C) or PCV2Δ-inoculated mice (D) for IFA analysis (bars, 50 μm). (E) Dot blot analysis in which mock- and wPCV2-infected PK-15 cells were lysed with the CytoBuster protein extraction reagent. After treatment with or without reducing loading buffer, the lysates were dotted onto a nitrocellulose membrane, which was incubated with the anti-ORF4 2D3 MAb followed by HRP-labeled goat anti-mouse IgG. Well 1, nonreduced mock-infected PK-15 cell lysate; well 2, nonreduced PCV2-infected PK-15 cell lysate; well 3, reduced wPCV2-infected PK-15 cell lysate; well 4, reduced mock-infected PK-15 cell lysate.