EMSA analysis of NF-κB and AP-1 in wild-type and PKCθ^–/– T cells. (A) Nuclear extracts were prepared from purified mature CD3⁺ wild-type and PKCθ^–/– T cells, stimulated for 16 hours with medium alone or plate-bound anti-CD3 plus soluble anti-CD28, as indicated. Gel mobility shift assays were performed using radiolabeled probes containing either (A) NF-κB or (B-C) AP-1 binding site sequences. (C) To study potential additive effects in PKCθ-deficient T cells, 5-fold more PKCθ^–/– nuclear extracts had been used. The specificity of p50 NF-κB as well as fos was confirmed by supershifting the electrophoretic mobility shift with antibodies, as indicated by the arrow. Experiments were repeated at least 3 times with similar results.