Identification of perturbations in this pathway in the context of human disease is worthy of further study. Supporting Information Figure S1 PMA plus ionomycin-induced IL-2 mRNA is stable in TNF-treated cells. Control and TNF-treated 11A2 cells were stimulated for four hours with P+Ilow prior to addition of actinomycin D 10 mg/ml. Cells were harvested at the times indicated and RNA extracted and analysed for IL-2 mRNA by ribonuclease protection assay. (A) Phosphorimage of protected RNA species; (B) IL-2 mRNA normalised to L32 mRNA and expressed as% peak levels (at time of addition of actinomycin D) for control and TNF-treated cells. Mean +/2 SD, 3 experiments. (PDF) Figure S2 Constitutive nuclear NFAT1 in 11A2 cells. Dephosphorylation and nuclear translocation of NFAT1 in control and TNF-pre-treated cells. 11A2 cells were cultured with or without TNF 2.5 ng/ml for 8 days before restimulation with PMA 10 ng/ml and ionomycin 50 ng/ml (P+I)low for the times indicated. Nuclear and cytoplasmic extracts were prepared and equivalent amounts of proteins assayed for the presence of NFAT1 by immunoblot. This filter was subsequently re-probed for NFAT2 and loading controls (see Figure 1D). Representative of three similar experiments. (PDF) Figure S3 Attenuation of DNA-binding for AP-1 proteins and NFAT2 in TNF-treated cells. Control and TNF-treated cells were stimulated for 4 hours with P+Ihigh. Nuclear extracts were incubated with 32P-labelled (A)(B) AP-1 or (C) NFAT/AP-1 oligonucleotide, with or without supershifting antibodies for (A) fos proteins, (B) jun proteins or (C) NFAT2. Protein-bound oligonu- cleotide was visualised by phosphorimaging after gel electropho- resis. (PDF) Figure S4 NF-kB and IkB in input cytosol and post- immunoprecipitate supernatants. Control and TNF-treated References 1. Huxford T, Huang DB, Malek S, Ghosh G (1998) The crystal structure of the IkappaBalpha/NF-kappaB complex reveals mechanisms of NF-kappaB inacti- vation. 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