FIG. 4.

The virus-binding activity shed from HeLa cells is a VLDLR fragment. HeLa suspension cells (8 × 10⁸) were incubated with 20 ml of PBS for 40 min at 37°C. The cells were pelleted, and the S80 supernatant was applied onto a GST-RAP affinity column. Fractions (0.5 ml) obtained upon elution with 1 M ammonia were collected and concentrated to 150 μl, and aliquots of 20 μl (corresponding to approximately 5 × 10⁷ cells) were analyzed by polyacrylamide gel electrophoresis on 10% gels, followed by ligand blotting with [³⁵S]methionine-labeled HRV2, with LDLR-specific IgY, and with rabbit antiserum against VLDLR, respectively. A 20-μl aliquot of the original unconcentrated sample (lanes S) and 20 μl of the flowthrough (lanes F) were also analyzed. The positions of marker proteins run on the same gels are also indicated. hVLDLR, human VLDLR.