Figure 2
rAd-mediated expression of rEDIII-4/2 protein as aGFP fusion. (A) Detection of rAd-mediated bivalent antigen expression by fluorescence microscopy. HEK 293 cells were either infected with rAd-C (panel i), or rAd-Bg (panel ii-iv) for 24 hours and examined either directly for GFP fluorescence (panels i and ii) or analyzed by IFA, using either mAb 3H5 (panel iii) or MAB8704 (panel iv), as the primary antibody in conjunction with rhodamine-conjugated anti-murine IgG as the secondary antibody. (B) Immunoprecipitation of the bivalent protein from infected HEK 293 cells. Cells were infected with rAd-C (lane 2), a rAd expressing DEN-2 E (lane 3 [ref. 45]) or rAd-Bg (lane 4) viruses. Infected cells were metabolically labeled with [35S]-methionine, lysed, immunoprecipitated with 3H5 mAb, and analyzed on a denaturing 15% polyacrylamide gel. Pre-stained protein markers were run on the same gel (lane 1); their sizes (in kDa) are indicated on the left. The arrows on the right show the positions of the bivalent protein (upper) and the E protein (lower).