Figure 3.
Intracellular Localization of Orf7a
(A) Flow cytometry analysis of protein expression levels on 293T cell transfectants. Direct comparison of orf7a-GFP with its cytoplasmic tail mutant, orf7aAA-GFP, in which Lys103 and Lys105 were replaced by Ala. The extent of transfection is revealed by GFP fluorescence. Both intact and saponin-permeabilized cells were stained with the anti-orf7a mAb 2E11. Significantly more anti-orf7a mAb staining was seen in the permeabilized cells, suggesting that the majority of the protein was intracellular.
(B) The same cDNA constructs were introduced into Vero cells and examined by confocal microscopy. Orf7a-GFP colocalizes best with the Golgi marker Golgin 97 (see Golgin 97 merge in upper row), while orf7a-AA-GFP colocalizes best with the ER resident protein calnexin (see calnexin merge in lower row). In all cases, the nuclei were counterstained blue with Topro, and the GFP fluorescence appears green. Optical slices were reconstructed into a three-dimensional image to show colocalization before compression into a two-dimensional representation.