FIG. 7.
Expression of E6 alters the binding between apoptotic signaling-protein partners. (A) E6small/E6* does not interfere with procaspase 8 homodimer formation. Lysates of both the parental U2OS cells and the cells expressing only E6small/E6* (U2OSE6S9) were separated by stepwise centrifugations as described in Materials and Methods. The material in each of the indicated fractions was then separated by SDS-PAGE, transferred to a membrane, and probed using a monoclonal antibody directed against caspase 8. (B) E6large, but not E6small/E6*, inhibits procaspase 8-procaspase 8 and procaspase 8-FADD binding. Cells (7 × 104) stably transfected with the antisense version of HA-E6 (U2OSE6AS), HA-E6large (U2OSE6L12), or HA-E6small/E6* (U2OSE6S9) were plated in wells of a 24-well plate and transfected with the indicated combinations of plasmids, along with a reporter plasmid coding for firefly luciferase under the control of the 5× GAL4 binding site. To control for transfection efficiency, pcDNA3-GFP was cotransfected with the above-mentioned plasmids. Binding was measured by monitoring expression of the luciferase gene, and the results are presented as a ratio of the signal measured for the indicated plasmid combination divided by the signal measured for GFP. The error bars indicate standard deviations. **, 0.99 level of confidence.