PKC-θ is required for NFAT and AP-1 activation. (A) IL-2–cultured PKC-θ^−/− and WT NK cells were stimulated with PMA (50 ng/mL) and ionomycin (0.5 μg/mL), and transcription of IFN-γ was analyzed by RT-PCR. (B) IL-2–cultured PKC-θ^−/− and WT NK cells were stimulated with PMA (50 ng/mL) and ionomycin (0.5 μg/mL). Cells were lysed and then analyzed by immunoblotting with anti-IκBα, anti–phospho-p38, ERK, JNK, c-Fos, and NFAT1-Ser54. As a loading control, membranes were probed with anti-actin antibody. (C) IL-2–cultured PKC-θ^−/− and WT NK cells were stimulated as in panel B, and then nuclear extracts were analyzed by EMSA using probes containing NFAT or AP-1 binding sites. Results are representative of 3 independent experiments.