Figure 4

Potentiation of NF-κB DNA binding and transcriptional activity by cAMP. (A) Reh cells were treated with or without forskolin (80 μM) for 30 min before irradiation (10 Gy). Cells were harvested at the indicated times after IR and nuclear extracts were prepared. The binding activity of p65 in the nuclear extracts was then determined by the p65 TransAM assay (n = 4). *p < .01, **p < .05 relative to cells treated with IR only. (B) Reh cells were transfected with a plasmid encoding the luciferase gene under 3 repeats of a NF-κB consensus binding site. After 24 h, cells were cultured in the presence or absence of forskolin (80 μM) for 30 min before exposure to IR (10 Gy). At the indicated times after IR, cells were harvested and the luciferase activity was measured as described in Materials and methods (n = 4). *P < .01 relative to cells treated with IR only. (C) Reh cells were transfected as in B. After 24 h, cells were cultured in the presence or absence of forskolin (80 μM), 8-CPT-cAMP (200 μM) or 8-pCPT-2'-O-Me-cAMP (400 μM) for 30 min before exposure to IR (10 Gy). Cells were harvested at 4 h after IR and luciferase activity was measured as described in Materials and methods (n = 4). *P < .01, **P < .05 relative to cells treated with IR only. (D) Freshly isolated splenocytes from 3 × κB-luc transgenic mice were cultured in the presence or absence of forskolin (80 μM) for 30 min before exposure to IR (10 Gy). After 2 h, luciferase activity was measured as described in Materials and Methods. The boxes show the median, upper and lower quartile, and the whiskers show the range of values (n = 9, *P < .05 by Wilcoxon signed rank test when compared with cells treated with IR only). The outlier value (○) stems from one mouse.