FIG. 9.

The ORF 3 protein associates with GV ORF 2 VLPs but not rotavirus VP2/6 VLPs. (A) GV VLPs (25 μg) were analyzed in an SDS–15% polyacrylamide gel. Western blot analysis was first performed using the rabbit ORF 3 C-terminal peptide antiserum. (B) The blot was then stripped in ECL stripping buffer as specified by the manufacturer (Amersham) and reprobed with the rabbit anti-rGV serum (1:500); the rGV ORF 2 and degradation products are detected by this serum. (C) Rotavirus VLPs (25 μg) were loaded for separation in an SDS–15% polyacrylamide gel and analyzed by Western blotting using the ORF 3 C-terminal peptide antiserum. Lane designations are as follows: rGV 2, GV ORF 2 VLPs; rGV 2/NV 3, dually infected GV ORF 2 and NV ORF 3 VLPs; rNV 2+3, NV ORFs 2+3 VLPs; VP2/6/3, dually infected rotavirus 2/6- and NV ORF 3 VLPs; VP2/6, rotavirus 2/6-VLPs. The open arrowhead designates where the ORF 3 protein would migrate if present. The asterisks show the location of rotavirus VP6 that is detected nonspecifically by the secondary antibody because of the large amount of protein on the gel.