Figure 2

Degradation of IκBα and nuclear translocation of p65 are induced by PRRSV Nsp2.

(A) HeLa cells were transfected with the indicated amount (0, 1, 2, 4 μg) of Nsp2 expression plasmid. Cytoplasmic and nuclear extracts were prepared at 48 h post-transfection and subjected to western blot analysis with antibodies specific for endogenous IκBα, p65 or p-p65. Mouse anti-HA was used to confirm the expression of HA-conjugated Nsp2 protein. Anti-β-actin was included as a control for sample loading. Western blot analyses were repeated in two independent experiments with similar results and a representative blot was shown. (B) HeLa cells were co-transfected with RFP-tagged Nsp2 expression plasmid (pDsRed-Nsp2) and the EGFP-tagged p65 expression plasmid (pEGFP-p65). Cells co-transfected with pDsRed-C1 and EGFP-tagged p65 served as a negative control. Cells were fixed and permeabilized at 36 h post-transfection. Cellular nuclei were counterstained with 1 μg/mL of DAPI (4',6-diamidino-2-phenylindole, dihydrochloride). Nuclear translocation of NF-κB was observed under an LSM-510 Meta confocal fluorescence microscope. Scale bar represents 5 μm.