Effect of IL-12 on STAT4 activation. (A) Western blot. B cells were activated with IL-12 for 15 and 45 minutes and then lysed. Western blotting was performed with whole-cell lysates by using antiphospho-STAT4. Membranes were reprobed with anti-STAT4. To correct for variations in the amount of loaded proteins, values are expressed as pSTAT/STAT ratios. Phospho-STAT/STAT levels were determined by densitometric analysis, including correction for background (NIH Image software). Results are expressed as -fold increases versus untreated cells. (B) Confocal microscopy of primary B cells. B cells were activated with IL-12 for 15 minutes. They were then fixed, permeabilized, and stained with anti-STAT4 (i-ii; green) and propidium iodide (nuclear staining, red). Yellow spots indicate nuclear STAT4 localization. (Bi) Untreated cells; (Bii) IL-12-activated cells. The small squares show STAT4 staining (green) without superimposition. In control experiments, anti-STAT4 was replaced by a rabbit IgG isotypic control. (Biii) Isotypic control of IL-12-treated cells. (C) Confocal microscopy of the RPMI 8866 cell line. Cells were extensively washed then treated with neutralizing anti-IL-12 for 1 hour. Cells were fixed, permeabilized, and stained as described. (Ci) Untreated cells; (Cii) Anti-IL-12-treated cells; (Ciii) Isotypic control of untreated cells. For panels A to C, similar results were obtained in 2 other experiments. Original magnification, × 63.