cells under conditions favouring T cell expansion. We therefore focussed our studies of IkBe/c-Rel-dependent regulation of lymphocytes on B cells. Increased basal nuclear c-Rel and enhanced BCR- stimulated c-Rel translocation in na¨ıve IkBe2/2 B cells Splenic B lymphocytes were obtained from three each of age- and sex-matched WT and IkBe2/2 mice. Cells were .93% B220+ by flow cytometry after CD19+ positive selection (not shown). First, we determined levels of c-Rel, p65 and IkB isoforms in nuclear and cytoplasmic extracts of B cells cultured in the presence or absence of stimulatory anti-IgM for up to one hour. Basal levels of nuclear c-Rel relative to nuclear actin were higher in na¨ıve B cells from three IkBe2/2 mice compared to those of WT (Figure 7, left side panel 1). Stimulation for 0.5 hour with anti-IgM induced further nuclear translocation of c-Rel in WT and IkBe2/2 B cells and this translocation was much stronger in B cells from IkB2/2 mice (Figure 7, right side panel 1). After 1 hour’s stimulation, nuclear c-Rel in WT B cells approached levels seen for IkBe2/2 B cells. Upon re-probe of the same blot, basal nuclear p65 was not detected in WT or IkBe2/2 B cells, and p65 was only faintly detectable in WT B cell nuclear extracts after 1 hour’s BCR stimulation (Figure 7, panels 2). p65 was strongly detected in one of the IkBe2/2 B cell nuclear extracts at this time-point, but the sample appears to be overloaded (see corresponding actin signal in panel 3). Cytosolic expression of p65 was similar in WT and IkBe2/2 Regulation of c-Rel by IkBe in Murine Lymphocytes B cells throughout (Figure 7, panels 5). IkBe was detected in WT but not IkBe2/2 B cell cytosols and was degraded following BCR ligation (Figure 7, panels 6). Nuclear expression of c-Rel was enhanced in the absence of IkBe even though IkBa expression was higher in IkBe2/2 than in WT B cells (Figure 7, panel 6), as had been the case for T cells. These data strengthen the case for a non-redundant role of IkBe in the regulation of c-Rel in lymphocytes. Enhanced survival of na¨ıve IkBe2/2 B cells is characterised by increased basal expression of CD40 and higher expression of BAFF-R ex vivo To test whether enhanced nuclear translocation of c-Rel affected lymphocyte survival, splenic B cells from 3 WT and 3 IkBe2/2mice were maintained for up to 72 hours either without antigen receptor stimulation (Figure 8 A–C), or in the presence of anti-IgM and IL-4 (Figure 8, D-F). TNF (20 ng/ml) was included in some cultures to test whether it affected expression of IkBe, or behaviour of WT B cells ex vivo. Viability of cells was determined by flow cytometry as the percentage of B220+ cells in the live cell gate and in order to explore the mechanism of enhanced survival, expression of c-Rel dependent pro-survival receptors CD40 and BAFF-R on live B220+ cells was determined at the same time. In the absence of BCR ligation, mean viability of B cells fell rapidly between d0 and d1, and further from d1 to d2. Viability at both d1 and d2 was significantly higher in IkBe2/2 B cells, compared to WT cells (Figure 8B). Basal expression of CD40, and Figure 7. Increased basal and BCR-inducible nuclear c-Rel in IkBe2/2 splenic B cells. Splenic B cells were isolated by CD19 positive selection from three each of IkBe+/+ and IkBe2/2 mice. (A) Cells were rested for 2 hours then stimulated with anti-IgM F(ab’)2 fragment 10 mg/ml for the times indicated at 107 B cells/ml per condition in a 12-well plate. There were insufficient cells from IkBe2/2 mouse 2, following selection, to carry out stimulation of its B cells. Nuclear and cytoplasmic extracts from resting and stimulated cells were analysed for nuclear NF-kB and IkB by immunoblot. Nuclear and cytoplasmic c-Rel were normalised to actin and expressed relative to values for B cells of IkBe+/+ mouse 1 (mouse numbers allocated randomly), as indicated. doi:10.1371/journal.pone.0024504.g007 PLoS ONE | www.plosone.org 12 September 2011 | Volume 6 | Issue 9 | e24504