FIG. 3.

Functional characterization of sCAR fusion proteins. (A) Inhibition of Ad binding. ³H-labeled Ad was preincubated with different amounts of either sCAR-His₆ or sCAR-EGF, and then ³H-Ad/sCAR-ligand complex samples (10⁵ cpm) were mixed with 293 cells (10⁶ cells per aliquot) and allowed to bind at 4°C. Cell-bound radioactivities were determined as described in Materials and Methods. Data are presented as the percentage of input ³H-Ad bound after washing and calculated as the cumulative mean ± SD of triplicate determinations. Error bars depicting SDs are smaller than the symbols. (B) Inhibition of Ad-mediated gene transfer. Recombinant Ad vector AdCMVLuc, expressing the firefly luciferase reporter gene, was preincubated with various amounts of either sCAR-His₆ or sCAR-EGF. Monolayers of 293 cells were then exposed to AdCMVLuc/sCAR-ligand complexes and assayed for luciferase activity as described in Materials and Methods. Gene transfer indices were calculated from the ratio of the mean luciferase activity documented in cells infected with either AdCMVLuc/sCAR-EGF or AdCMVLuc/sCAR-His₆ to those treated with AdCMVLuc alone. Each point represents the cumulative mean ± SD of triplicate determinations. Some error bars depicting SDs are smaller than the symbols.