EMSA and nuclear translocation analysis of NF-AT in wild-type and PKCθ^–/– T cells. 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 in combination with the PDE4 LMWI, the Sp-8-Br-cAMP, and the Epac-specific activator (8-pCPT-2′-O-Me-cAMP), as indicated. In EMSA (A,B,D), the specificity of NF-ATc was confirmed by supershifting the electrophoretic mobility shift with antibodies, as indicated by the arrow. To study potential additive effects using EMSA methodology in PKCθ-deficient T cells, 5-fold more PKCθ^–/– nuclear extracts had been used (B), as PKCθ gene ablation already strongly reduced NFAT levels (A). Activation-induced translocation of NF-AT or phosphorylation of CREB was determined by immunoblotting of nuclear extracts (C,E) for NF-ATc, (p)CREB and panCREB, respectively, as indicated. Experiments were repeated at least 3 times with similar results.