IL-12 promoter-driven luciferase assay. Human IL-12 p40 (A) and p35 (B) promoter/luciferase reporter constructs were generated by ligating the human p40 (−1.3 kb/+56 bp) promoter and p35 (−1.5 kb/+3 bp) promoter fragments upstream of the luciferase gene in pGL3-Basic vector. RAW267.4 cells were transiently transfected and then stimulated with murine recombinant IFN-γ followed by LPS in the presence or absence of 100 nM STA-5326 or 1 μM STA-5392, a structurally related inactive compound in duplicate. Cells were cotransfected with pCMVβ, a vector in which the constitutively active CMV promoter drives expression of β-galactosidase, for the monitoring of transfection efficiency. The relative luciferase activity is the mean ratio of luciferase activity in the treated group relative to luciferase activity in the nontreated group after normalization with the β-galactosidase value.