Regulation of c-Rel by IkBe in Murine Lymphocytes oligonucleotide was detected at 2 hours’, and increased after 4 hours’ stimulation with PMA and ionomycin in control cell extracts (Figure 3A, lanes 7 and 9). Two inducible protein/DNA complexes (upper complex I, lower complex II) were observed. At these time points, which correspond to those for induction and nuclear translocation of c-Rel by PMA and ionomycin, the intensity of signal due to both complexes, particularly complex I, was lower in TNF-treated relative to control cell samples (Figure 3A, compare lanes 7 and 8, and 9 and 10). Analysis of NF-kB in complexes I and II following 4 hours’ stimulation of cells, using subunit-specific Figure 3. Transcription factor DNA binding and activity are suppressed by TNF. Activation of pCD28RR partially restored by exogenous c- Rel. Control and TNF-treated 11A2 T cells were activated with (P+I)high. Nuclear extracts were incubated with (A) 32P-labelled NF-kB consensus oligonucleotide or cold competitor probe or (B) 32P-labelled NF-kB oligonucleotide with or without NF-kB supershifting antibodies, before non- denaturing gel electrophoresis. Protein-bound oligonucleotide was visualised by both phosphorimaging and exposure of the dried gel to photographic film. Images are representative of three experiments. Control and TNF-treated cells were transfected with reporter plasmids (10 mg) expressing firefly luciferase under control of (C) pAP-1, (D) pNFAT/AP-1, (E) pNF-kBconsensus, (F) pCD28RR(NF-kB/AP-1) promoters, and stimulated as above for 6 hours. Firefly luciferase expression relative to pSV40-driven renilla luciferase from a co-transfected plasmid (0.2 mg) is shown (mean +/2 SEM for 9, 7, 4 and 5 experiments, respectively). (G) Summary of inducible promoter activities in TNF-treated cells as a percentage of induction in control cells, for all experiments. (H) Control and TNF-treated 11A2 cells were cotransfected with 2 mg of empty vector or c-Rel expression plasmid and pCD28RR reporter plus renilla luciferase plasmids prior to stimulation as for F. The dotted line represents stimulated expression in cells transfected with empty vector (mean +/2 SEM, n = 4 experiments). doi:10.1371/journal.pone.0024504.g003 PLoS ONE | www.plosone.org 7 September 2011 | Volume 6 | Issue 9 | e24504