FIG. 2.

Characterization of the material shed from HeLa cells by Western analysis with specific antibodies. Material released from HeLa cells upon incubation with PBS for 40 min at 37°C was cleared and concentrated, and proteins were separated on a 4 to 12% polyacrylamide gradient gel (A) or on a 10% gel (B) under nonreducing conditions and electrophoretically transferred onto PVDF membranes. The membranes were incubated with ³⁵S-labeled HRV2, antisera, and monoclonal antibodies as indicated at the top of the panels. Virus was detected by autoradiography. Bound antibodies were detected with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Promega) diluted 1:5,000; detection of scFv7 was with the anti-myc antibody 9E10 (Stratagene), followed by HRP-conjugated rabbit anti-mouse IgG (Southern Biotechnology) diluted 1:5,000. IgG-C7 was detected with HRP-conjugated rabbit anti-mouse IgG (Southern Biotechnology) diluted 1:5,000. HRP activity was revealed with the chemiluminescence substrate Supersignal (Pierce). Control HeLa cell membranes were prepared as described previously (17). The positions of marker proteins run on the same gels are indicated. memb., HeLa membranes; sup., material shed from HeLa cells.