Figure 4. Association of IkBe with newly synthesised c-Rel and p65/c-Rel complexes in TNF-treated cells. Control and TNF-treated 11A2 cells were restimulated with (A) (P+I)high, (B) P+Ilow, for the indicated times. Nuclear and cytosolic extracts were prepared. (A) IkBs a, b and e were immunoprecipitated from 100 mg cytosolic protein and immunoblotted for associated c-Rel and p65. (B) p65-containing complexes were immunoprecipitated from 100 mg cytosolic protein and immunoblotted for associated IkB. Blots were probed and reprobed for the indicated proteins and those shown are representative of more than 3 experiments at both concentrations of P+I. doi:10.1371/journal.pone.0024504.g004 p65 itself was not always pulled down with identical efficiency, as may be seen by variation in immunoprecipitated p65 signal compared to the signal in input cytosol (Figure 4B, panel 1 and Figure S4B, panel 1). All three IkBs associated with p65/c-Rel complexes. In resting and stimulated cells, the amounts of IkBs -a and -b associated with p65/c-Rel were similar for control and TNF-treated cell samples, once efficiency of the p65 pull-down was taken into account (Figure 4B, panels 3 and 4 – compare with panel1). However, at all time-points, more IkBe was associated with the p65/c-Rel complexes in TNF-treated than in control cell extracts even when expression of IkBe in control cells approached that of TNF-treated cells, at four hours (Figure 4B, panel 5: lanes 9 and 10, compare with Figure S4B panel 5). Taken together, these experiments indicate that IkBe sequesters newly-synthesised c-Rel in propor- tion to its own expression levels, and exhibits persistent, enhanced association with p65/c-Rel complexes in TNF-treated cells. To examine whether regulation of IkBe by TNF was a general phenomenon, as opposed to a peculiarity of T cell hybridomas, we cultured murine B cell lines, including A20 and hybridoma SP2/0, as well as primary T cells of Balb/C and C57Bl/6J mice, in the presence or absence of TNF. However, we failed to document Regulation of c-Rel by IkBe in Murine Lymphocytes TNF-dependent upregulation of IkBe. Indeed, we observed very high levels of IkBe in primary T cell blasts, regardless of the presence of TNF. Therefore, in order to test our hypothesis – of selective regulation of c-Rel by IkBe in primary cells – and explore its functional consequences, we focussed our studies on lympho- cytes from IkBe-deficient mice. Increased basal nuclear c-Rel and reduced threshold for TCR-dependent proliferation in IkBe mouse T cells We looked first at c-Rel nuclear expression in murine primary T cell blasts, as a correlate of experiments in the 11A2 T cell line. Splenocytes and lymph node cells (LNC) from age- and sex- matched wild-type (+/+), heterozygote (+/2) or homozygous IkBe (2/2) mice were cultured for 48 hours in the presence of anti-CD3 and expanded for 6 days in IL-2. T cell blasts were then re-stimulated for up to 4 hours with plate-bound anti-CD3 and analysed by immunoblot for NF-kB and IkB proteins. We detected increased nuclear c-Rel in resting T cell blasts from both IkBe+/2 and IkBe2/2 mice when compared to IkBe+/+, and observed a negative correlation between IkBe gene dose and nuclear c-Rel (Figure 5A and B, panels 1, 0 h time-points). This difference in nuclear c-Rel was most marked for resting T cells and decreased with time of TCR-restimulation until, by four hours’ TCR ligation, similar amounts of nuclear c-Rel were detected for all genotypes (Figure 5A, panel 1). Relative band intensities due to c-Rel normalised to nuclear actin in resting T cells are summarised for one experiment in Figure 1B and for all three experiments in Figure 5C and Table S1. Tubulin was not detected in the nuclear extracts indicating that they were free of cytosolic contamination (Figure 5A and B, panels 3). Nuclear p65 was detected at lower levels overall in resting T cells than was c-Rel, and any increase due to IkBe-deficiency was not consistently observed (Figure 5A and B, panels 2 and Table S1); nuclear p50 was not detected. We also observed increased basal IkBa expression in resting cells from IkBe2/2 mice (Figure 5A, panel 4, Figure 5D, and Table S1), as has been described [47]. c-Rel was detected in nuclei of resting IkBe2/2 T cells despite increased expression of IkBa and normal levels of IkBb, indicating that IkBe has a non-redundant role in the inhibition of c-Rel nuclear translocation. We wondered whether T lymphocytes would be primed for c-Rel-dependent upregulation of IL-2 in IkBe2/2 mice. We found that, although PMA and ionomycin- induced IL-2 expression showed an inverse correlation with IkBe gene dose (Figure 5E), the effect was not statistically significant, and there was no genotype-specific difference in IL-2 mRNA expression by qRT-PCR, in T cell blasts stimulated with plate- bound anti-CD3 (not shown). Further, there was no detectable genotype-dependent difference in the amount of IL-2 secreted following TCR-stimulation of na¨ıve splenocytes or LNC, or T cell blasts. By contrast, when we measured 3H-thymidine incorpora- tion by na¨ıve splenocytes and LNC in response to soluble anti- CD3, we found that IkBe+/2 and 2/2 cells appeared more sensitive to stimulation with concentrations of anti-CD3 of less than 20 ng/ml than did WT cells, but the effect was statistically significant only for IkBe2/2 cells (Figure 5F). Anti-CD28 alone had no stimulatory effect and did not co-stimulate at concentra- tions below 1 mg/ml. Increased basal thymidine uptake in IkBe2/2 cells and increased cellularity of IkBe2/2 lymph nodes Increased basal thymidine incorporation by IkBe2/2 and IkBe+/2 cells relative to WT cells was observed consistently for splenocytes, LNC and T cell blasts, and was statistically significant PLoS ONE | www.plosone.org 9 September 2011 | Volume 6 | Issue 9 | e24504