siRNA interference of IL-12 transcription and translation in vitro. J744A.1 cells were transfected with IL-12p40 siRNA for different periods (24–72 h). For the last 12 h of culture, cells were stimulated with LPS/IFN-γ or with PBS (-LPS, hereafter termed unstimulated). Expression of IL-12p40 (A) was determined by sqRT-PCR. Samples were normalized for β-actin expression (lower panel A). Control siRNA transfections included naked siRNA for IL-12p40, mutant siRNA for IL-12p40 and GFP (B). IL-12p70 protein expression was determined by ELISA (C). Data are representative of at least four independent experiments; IL-12p70 protein concentration is expressed as the mean (+/-SEM) from triplicate cultures (n = 3) on each occasion.