antibodies, revealed that complex II appears to contain chiefly p50 protein (Figure 3B, lanes 9 and 10), and complex I, p65, c-Rel and p50 (lanes 5–10). Markedly less c-Rel, p65 and p50 were detected in inducible complexes I and II derived from TNF-treated, as compared to control cell samples (compare lanes 4, 6, 8 and 10 with lanes 3, 5, 7 and 9). This difference in DNA-bound NF-kB was greatest for complex I, consistent with the more marked TNF- dependent reduction in nuclear c-Rel and p65 relative to p50 already observed. Maximal inducible AP-1 protein binding to radiolabelled probe was also greater in control compared to TNF-treated cell nuclear extracts. Subunit-specific supershift indicated modest reductions in DNA bound c-Fos and Jun B in TNF-treated cell extracts compared to controls, but little or no difference in the amounts of Fos B, Fra-2, c-Jun, or Jun D (Figure S3B, C; c-Jun not shown; Fra-1 not detected). EMSA and supershift with anti-NFAT2 antibody revealed similar amounts of NFAT2 in inducible NFAT/ AP-1 binding complexes from control or TNF-treated cell samples, despite reduced complex size for the latter (Figure S3D). However, since reduced NFAT/AP-1 complex size in TNF- treated cell samples was consistent in three experiments but the supershift worked well only in the assay shown, the discrepancy between antibody-supershifted and unshifted complex sizes may be unique to this particular experiment. NFAT1 which was constitutively nuclear in 11A2 cells and expressed to similar levels in control and TNF-treated cells (Figure S2) was not assayed for NFAT/AP-1 DNA binding. In parallel experiments, we tested the effect of reduced nuclear levels and attenuated DNA-binding of these transcription factors on the ability of cells to activate promoters containing consensus sequences for NF-kB, AP-1 and NFAT. The two composite sequences in pIL-2 which have been shown to be important for gene activation are an NFAT/AP-1 site at position 2274 to 2287 [27], and an NF-kB/AP-1 site also known as the CD28 response region (CD28RR) at position 2147 to 2163 from the transcrip- tion start site [28]; the non-consensus NF-kB-binding element in CD28RR (CD28RE) preferentially binds c-Rel-containing dimers in stimulated T cells [48]. We measured PMA and ionomycin- stimulated expression of firefly luciferase under control of pAP-1, pNFAT/AP-1, pNF-kB, or pCD28RR. Basal and induced firefly luciferase activities were expressed relative to co-transfected constitutive Renilla luciferase expression in the same cell lysate sample (see Methods). Transcriptional activation was suppressed in TNF-treated cells as compared to control cells, for all promoters tested (Figure 3C– 3F), by 55–60% for pAP-1 and pNFAT/AP-1, 80% for pNF-kB and 75% for pCD28RR (Figure 3G). These data correlate with the greater attenuation which we observed in nuclear expression and DNA-binding of NF-kB, particularly c-Rel, relative to NFAT and AP-1, in TNF-treated cells. We therefore tested the ability of exogenous c-Rel to overcome suppressed activation of pCD28RR in TNF-treated T cells by co-transfecting c-Rel expression vector, or empty vector, with firefly luciferase reporter and constitutive Renilla luciferase plasmids. Co-transfection of TNF-treated 11A2 cells with c-Rel restored inducible activation of pCD28RR to a level equivalent to that of control cells transfected with empty vector (Figure 3H). However, exogenous c-Rel led to a 4-fold increase in inducible pCD28RR activation for control as well as TNF-treated cells, implying firstly that c-Rel concentration is limiting in terms of activation of CD28RR in untreated 11A2 cells and, secondly, that reconstitution of factors other than c-Rel – such as AP-1 subunits – is required for maximal activation of CD28RR in TNF-treated cells. Regulation of c-Rel by IkBe in Murine Lymphocytes PMA and ionomycin-induced c-Rel associates with IkBa and IkBe; persistent and enhanced association of IkBe with p65/c-Rel in TNF-treated cells throughout stimulation Our data implied that TNF pre-treatment had inhibitory effects on all three transcription factor families required for optimal activation of the IL-2 promoter. However, we became most interested in the selective increase in IkBe expression, out of the IkBs, accompanied as it was by strong inhibition of c-Rel nuclear translocation, out of all NF-kB subunits tested, in TNF-treated cells. We decided to use this system to investigate the association of c-Rel and IkBe further. c-Rel has been shown to be chiefly associated with IkBb in na¨ıve, but with IkBa in TNF- and IL-1-stimulated, T cells [49]. However, the contribution of IkBe was not examined in that study. Biochemical analysis demonstrated that IkBe binds c-Rel and p65 hetero- or homodimers in preference to p65/p50, which tends to be bound by IkBa [8]. We therefore explored the composition of NF-kB/IkB complexes in TNF pre-treated cells following stimulation with PMA and ionomycin. We immunopre- cipitated IkBa, -b, or -e from equal amounts (100 mg) of cytosolic protein, and immunoblotted for associated NF-kB (Figure 4A; see Figure S4A (i) and (ii) for proportions of each protein in cytosol and post-IP supernatants). IkB proteins were precipitated efficiently by their individual antibodies, since little IkB was detected by immunoblot in post-IP supernatants (Figure S4A (i). However, p65 and c-Rel were still detectable in these post-IP supernatants (Figure S4B (ii)) suggesting that only a small proportion of either NF-kB co-precipitated with a single IkB isoform. In resting cells c-Rel was associated with IkBb (Figure 4A, panel 4) and p65 was associated with both IkBa and b (Figure 4A, panels 2 and 5). A small amount of c-Rel and p65 also co- immunoprecipitated with IkBe (Figure 4A, panels 7 and 8). The amounts of p65 or c-Rel associated with each IkB were reduced when the IkB was degraded, and increased as the IkB was resynthesised, following stimulation. Newly synthesised c-Rel, expressed after 2 hours, associated with IkBa and IkBe as well as IkBb (Panels 1, 4 and 7, 2–4 hour time-points). The amounts of p65 associating with IkBa and b, and the amount of c-Rel associating with IkBb, were similar for control and TNF-treated cell samples (Figure 4A, panels 2, 4 and 5). However, the amount of c-Rel associated with IkBe was greater in TNF-treated cell samples compared to controls up to 2 hours of stimulation (panels 7 and 9: 0–2 hour time-points). This difference was lost when IkBe expression in control cells reached that of TNF-treated cells (panels 7 and 9, 4 hour time-point). Thus the amount of c-Rel immunoprecipitating with IkBe reflected the expression level of IkBe. By contrast, association of newly-synthesised c-Rel with IkBa reflected the greater abundance of c-Rel in control compared to TNF-treated cells (Figure 4A, panels 1 and 3 and Figure S4A (i) and (ii)). In three experiments, it was difficult to obtain very clear bands for immunoprecipitated IkBs -b and -e, because the signal for these 45 kD proteins ran just in front of the heavy chain band of immunoprecipitating antibody in blots. We were unable to detect p50 in any co-immunoprecipitation experiment. We also immunoprecipitated p65, using directly-conjugated agarose beads, and blotted for associated IkB proteins. We were unable to immunoprecipitate c-Rel directly by a similar method, but found that a proportion of c-Rel co-precipitated with p65 (Figure 4B, panels 1 and 2). Blots of post-IP supernatants revealed efficient co-immunoprecipitation of IkBs with p65 (Figure S4C). PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e24504