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Nuclear extracts prepared from Cos-7 cells that are either transfected with T-bet or not and stimulated with PMA/ionomycin. Nuclear extracts are diluted to 0.156 μg/well and assayed using the TransAM T-bet Kit. The ratio of the signals from the stimulated over unstimulated cells must be above 4. Lot No. 29408001 was developed for 3 minutes. It gave a ratio of 120 (Figure 1). This ratio may vary depending on the level of T-bet activation in a given cell type. TransAM T-bet Kits are also tested for specificity in detecting T-bet activation. TransAM T-bet assays are performed in the presence of an excess of oligonucleotide containing a wild-type or mutated T-bet consensus binding site (Figure 2). At 40X excess, the wild-type oligonucleotide prevents T-bet binding to the probe immobilized on the plate. Conversely, the mutated oligonucleotide has no effect on T-bet binding.