Fig. 3. Coexpressed CAN inhibits IFN-1 gene expression in a plasmid dosage-dependent manner. HeLa cells were cotransfected at 37°C for 16 hours with a constant amount of either phuIFN-1 (open circles), pRSVluc-CTE (closed circles) or pCRRE (open triangles), and various amounts of pCG-HA-CAN that are shown on the x-axis. At 32 hours after the DNA addition, cytoplasmic lysates and culture supernatants were obtained to assay the IFN-1 concentrations (open circles) and luciferase activities (closed circles), or to analyse the p55^Gag expression by western blotting, as described in the Materials and Methods. The concentrations of IFN-1 and p55^Gag, as well as the luciferase activities measured, are given as the percentage protein concentrations or enzymatic activities relative to those from phuIFN-1, pCRRE or pRSVluc-CTE alone transfected HeLa cells. Values of a representative experiment of three independent transfection experiments are shown; mean±s.e.m. of triplicate samples. (Bars cannot be seen when they are smaller than the graph symbols.)