MAPK p38 is activated by IL-12 and IL-18 only in NK cells. (A) NK or T cells (3 × 10⁶), purified by negative selection from PBLs, were stimulated for 15 minutes with sodium arsenite (As), IL-12, and/or IL-18 (20 ng/mL). Lysates were prepared and Western blotted for phospho-p38 (top) or total p38 (bottom). (B) NK cells were purified by negative selection from PBLs, left untreated or stimulated with sodium arsenite (As) for 15 minutes, then stained with PE-conjugated anti-phospho-p38 antibody or its isotype control (left). In the right-hand panel, cells were treated with sodium arsenite and stained with PE-conjugated anti-phospho-p38 antibody in the presence of 0.1 mg/mL of a phosphorylated p38 peptide corresponding to the antigen (pep 1) or the corresponding nonphosphorylated peptide (pep 2). (C) NK cells were purified by negative selection from PBLs and left untreated or stimulated with IL-12 and IL-18 (20 ng/mL each) for 2, 3, or 4 hours in the presence of Brefeldin A. Intracellular IFN-γ and phospho-p38 content were assessed by 2-color flow cytometry. Note that methanol fixation is required for detection of phospho-p38 but is not optimal for detection of IFN-γ; therefore, expression of the cytokine is underestimated. (D) NK cells were purified by negative selection from PBLs, left untreated or treated with IL-12 and/or IL-18 (20 ng/mL) for the indicated times, and stained with PE-conjugated anti-phospho-p38 antibody. The isotype control is shown in gray in each panel, and estimated percentages of phospho-p38-positive cells are indicated.