Fig 1.

Detection of a transiently expressed recombinant His-ANDV NSs protein in Vero E6 cells. (A) Schematic representation of the plasmids used to express the recombinant tagged ADNV NSs and ANDV N proteins. The arrows indicate the initiation codon used to generate the recombinant proteins. BGH(A) corresponds to the bovine growth hormone polyadenylation sequences. (B) Plasmid pNSs-ANDV, expressing the recombinant His-ANDV NSs protein, was transfected in Vero E6 cells, and 48 h posttransfection total proteins were extracted. Shown is the expression of the recombinant His-ANDV NSs protein detected by Western blotting using a commercial anti-His monoclonal antibody (left) or the anti-NSs monoclonal antibody NS2-2B11/C2 (right). (C and D) Vero E6 cells transfected with plasmid pN-ANDV (C) or plasmid pNSs-ANDV (D) were fixed, and the expression of the His-tagged protein was detected by immunofluorescence using a commercial polyclonal anti-His antibody, anti-NSs monoclonal antibody NS2-5E7/D9, or a well-characterized anti-ANDV N monoclonal antibody (11, 30, 48). A goat anti-mouse fluorescein isothiocyanate (FITC)-conjugated antibody (green staining) and a goat anti-rabbit Alexa Fluor 555-conjugated antibody (red staining) were used as secondary antibodies. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue staining). (E) Vero E6 cells transfected with pNSs-ANDV, pN-ANDV, and the His-tagged irrelevant tagged protein upstream of N-ras (His-UNR) were fixed, and the expression of the His-tagged proteins was detected by immunofluorescence using a commercial anti-His monoclonal antibody, followed by an FITC-labeled anti-mouse antibody (green staining). The recombinant His-tagged ANDV NSs protein was specifically detected by the anti-NSs monoclonal antibody NS2-5E7/D9, followed by an FITC-labeled anti-mouse antibody (green staining). Cell nuclei were stained with DAPI (blue staining). For all panels in the figure, size bars corresponds to 20 μm.