Down-regulation of NF-κB DNA binding activity by RAD in L540cy and Karpas 299 cells. (A) The HL cell line L540cy was incubated with 0 nM (▪) to 10 nM (□) RAD for 24, 48, and 96 hours. Cell counts are given as the mean of 3 independent experiments. Counts of RAD-treated lymphoma cells are given relative to counts of untreated cells, which were set arbitrarily at 100%. (B) L540cy and Karpas 299 cells were treated with 0 nM to 10 nM RAD for indicated times. Whole cell extracts were analyzed by EMSA for NF-κB DNA binding activity. Free DNA probe is not shown. n.s. indicates nonspecific. (C) L540cy cells were transfected with an NF-κB-dependent reporter construct (6NF-κBtkluc) and transcriptional activity of NF-κB was detected 24 and 48 hours after treatment with 0 nM (▪) to 10 nM (□) RAD. Luciferase counts are given relative to control-treated L540cy cells, which are set arbitrarily at 1. Luciferase activity was determined for triplicate experiments. (D) Protein expression levels p50 and p65 in response to RAD analyzed by Western blot analysis in L540cy cells. (E) Protein and total mRNA were prepared after indicated times and analyzed for expression of IκBα by Western and Northern blotting in L540cy cells. Error bars indicate SD.