FIG. 9.
Coexpression of the ORFs encoding the GP2b, GP3, and GP4 proteins. EAV ORF2b, -3, and -4 were expressed in BSR T7/5 cells alone or together, as indicated at the top of the autoradiographs, by using the vaccinia virus-T7 RNA polymerase transfection system. The cells were pulse-labeled for 30 min with l-[35S]cysteine starting at 4.5 h p.i. (A) Cell lysates were subjected to immunoprecipitations with the αGP2b serum (upper panel) or the αGP4 serum (lower panel). The immunoprecipitates were treated (lanes +) or mock treated (lanes −) with endo H. As a control, lysates of cells that expressed only ORF2b, ORF3, or ORF4 were mixed in a 1:1:1 ratio and incubated with the αGP2b serum (lanes 234 mixed). The samples were analyzed in SDS-15% PAA gels under nonreducing conditions. For comparison, EAV-infected BHK-21 cells (lanes V) were pulse-labeled for 30 min with l-[35S]cysteine starting at 8.5 h after infection and further processed as described above. (B) Alternatively, immunoprecipitates (IP) prepared with the αGP2b (lanes 2) or αGP4 (lanes 4) serum in the presence or absence of 5 mM DTT were analyzed by SDS-PAGE under reducing conditions. Glycosylated protein species are indicated in uppercase letters and/or closed with symbols. Deglycosylated protein species are indicated in lowercase letters and/or with open symbols. The numbers on the left are the molecular masses, in kilodaltons, of marker proteins analyzed in the same gel. Ab, antibody.