The recombinant adenoviruses encoding murine p35 and/or p40 subunits express biologically active IL-12. (A) HeLa cells were transduced with Ad.GFP, Ad.p40, or Ad.IL-12 at the indicated MOIs. Cells or culture media were harvested 48 h after transduction. Cleared lysates of cells transduced with Ad.GFP or Ad.p40 were immunoblotted with anti-IL-12 antibody (left panel). The culture media from HeLa cells transduced with Ad.IL-12 or Ad.GFP were immunoprecipitated with anti-IL-12 antibody. The immunoprecipitates were dissolved in SDS sample buffer without reducing agent and subjected to SDS-PAGE, followed by immunoblotting with anti-IL-12 antibody (right panel). Molecular size markers in kilodaltons are shown at the left. The positions of p40, p35–p40 heterodimer (p70) and p40 homodimer (p80) are indicated by arrows. (B and C) HeLa cells were mock-transduced or transduced with either a 1:1 (pfu ratio) mixture of Ad.p35 and Ad.p40, or Ad.IL-12, or Ad.GFP, at the indicated MOI. Splenocytes from naïve mice were incubated with the culture supernatants for 24 h. ELISA was performed to determine the IFN-γ levels after incubation. Data are means ± SD of two independent experiments, each performed in duplicate.