Regulation of c-Rel by IkBe in Murine Lymphocytes Figure 2. TNF pre-treatment inhibits subsequent induction and nuclear expression of NF-kB and upregulates IkBe. 11A2 cells were cultured with or without TNF 2.5 ng/ml for 8 days before restimulation with PMA 10 ng/ml and ionomycin 50 ng/ml or TNF 50 ng/ml for the time- points indicated. Nuclear and cytoplasmic extracts were prepared and equivalent amounts of protein assayed for the presence of NF-kB transcription factors (A, B) and IkB isoforms (C, D) by immunoblot. Each blot represents at least three similar experiments, probed then reprobed for the indicated proteins. doi:10.1371/journal.pone.0024504.g002 at all time-points, compared to control cells (panel 5). Thus, pre- treatment of 11A2 cells with TNF also inhibited TNF-R-induced NF- kB nuclear translocation, most markedly that of c-Rel. These data suggested that the effects of TNF pre-treatment on NF-kB activation were not restricted to TCR/CD28-dependent pathways involved in IL-2 gene expression. Rather, TNF attenuated NF-kB activity by modulating a receptor-distal factor common to TCR- and TNF-R- dependent NF-kB activation, IkB being a good candidate. We therefore examined inducible degradation of IkBs -a, -b and -e by PMA and ionomycin (Figure 2C) or TNF re-stimulation (Figure 2D), in control and TNF-treated cells. TNF pre-treatment led to increased basal levels of IkBe, but not of -a or -b (Figure 2C, D: compare first and second lanes). IkBs -a and -b were rapidly degraded to similar degrees in control and TNF-treated cells in response to either stimulus (Figure 2C, D; panels 1 and 2) and were re-expressed after 1–2 hours (a), or 2–4 hours (b), of stimulation, re-expression of IkBb being more marked in control than TNF-treated cells. By contrast, IkBe was present at increased levels in TNF-treated compared to control cells for stimulations of up to 2 hours. IkBe also appeared resistant to PMA and ionomycin-induced degradation (compare 0.5 and 1 hour time- points of control and TNF-treated cells, Figure 2C, D, panels 3), and returned to unstimulated cell levels more rapidly in TNF pre- treated than in control cells (compare 0 and 1 hour time-points of control and TNF-treated cells, Figure 2C, D; panels 3). Thus, both inhibition of nuclear translocation of NF-kB, particularly of c-Rel, and sustained, increased expression of IkBe in TNF pre-treated cells were evident upon acute activation TCR and TNF-R pathways. Transcription factor DNA binding and activity are suppressed by TNF: transcriptional activity partially restored by exogenous c-Rel We used electrophoretic mobility shift assay (EMSA) to test whether reduced nuclear levels of NF-kB, AP-1 and NFAT proteins in TNF-treated 11A2 cells were reflected by attenuated DNA- binding. Inducible binding to radiolabelled NF-kB consensus PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e24504