Regulation of c-Rel by IkBe in Murine Lymphocytes Figure 5. Increased basal nuclear c-Rel, priming for IL-2 expression, and enhanced sensitivity to TCR stimulation in IkBe2/2 T cells. Splenocytes and LNCs from WT C57BL/6J (IkBe+/+, filled circles) mice, or mice hetero- (+/2, half-filled circles) or homozygous (2/2, open circles) for IkBe deletion, were cultured with anti-CD3 for 48 hours, washed, then stimulated with IL-2 at day 2 and day 5. On day 7, cells were washed and restimulated, or not, with plate-bound anti-CD3 for up to 4 hours. Extracts from resting and stimulated cells were analysed for (A) nuclear and cytosolic NF-kB and IkB – a four-hour time-course is shown – (B) nuclear NF-kB – a two hour time-course representative of three experiments is shown, with nuclear c-Rel and p65 normalised to actin and expressed relative to values for resting IkBe+/+ T cells as indicated. (C) Relative nuclear expression of c-Rel (mean +/2 SD, n = 3 experiments). (D) Cytosolic IkB for experiment shown in (B), with IkBa and -b normalised to actin and PLoS ONE | www.plosone.org 10 September 2011 | Volume 6 | Issue 9 | e24504