Enhanced NFκB in nuclear extracts of mouse T cells after exposure to ionizing radiation. T cells, enriched by either negative depletion or positive selection, or unfractionated splenocytes were irradiated at a dose rate of 5 Gy/min to 7.5 Gy or 30 Gy. Irradiated cells were cultured in RPMI + 10% FCS at 4°C or 37°C for 6 hours. Nuclear extracts were prepared and the relative level of nuclear NFκB determined by incubating 10 μg of nuclear protein with a ³²P-labeled oligonucleotide containing NFκB binding sequence. The far left lane labeled “NC” contained water with no nuclear extract; lane “PC” contained a Hela cell nuclear extract (positive control for NFκB) incubated with the³²P-labeled oligonucleotide alone; “PC + SC” contained a combination of the Hela cell nuclear extract, the³²P-labeled oligonucleotide, and an excess of the unlabeled specific oligonucleotide competitor; and “PC + NSC” contained a combination of the Hela cell nuclear extract, the³²P-labeled oligonucleotide, and an excess of an unlabeled nonspecific oligonucleotide. The next 3 lanes from the left contained nuclear extracts of unfractionated splenocytes irradiated at 0 Gy, 7.5 Gy, or 30 Gy then incubated at 37°C. The mean increase in the upper NFκB band of nonirradiated T cells compared with T cells irradiated to 7.5 Gy at 37°C was 4-fold (n = 5 EMSA from 2 separate isolations of T cells). Three lanes contained nuclear extracts of T cells enriched by negative selection irradiated at 0 Gy incubated at 37°C, 7.5 Gy incubated at 37°C, or 7.5 Gy incubated at 4°C. Two lanes on the right contained nuclear extracts of T cells enriched by positive selection irradiated at 0 Gy incubated at 37°C, or 7.5 Gy incubated at 37°C.