Reduced NF-κB activity in c-FLIP S -Tg T cells. (A) Purified T cells from NF-κB-luciferase mice (NF-κB-luc) or NF-κB-luc/c-FLIP S double transgenic mice (c-FLIP S -Tg x NF-κB-luc) were stimulated with anti-CD3/anti-CD28 for the days indicated. *Results were statistically significant on Day 2 (p=0.021), Day 3 (p=0.008), and Day 3 (p=0.015) by paired t-test. (B) Immunoblot of whole cell lysates from purified T cells from wild-type (WT) or c-FLIP S -Tg (Tg) mice using antibodies specific for p50, RelA, c-Rel, and actin. (C) Immunoblot analysis of anti-CD3/CD28-activated T cells for phospho-IκBα versus total IκBα, and phospho-IKKα/β versus total IKKα/β. Control activation was with PMA plus ionomycin (P+I) for 60 min. (D) EMSA for NF-κB binding of nuclear lysates from purified anti-CD3/CD28-activated T cell. Phosphoimager analysis of the same assay was quantified for the p65/p50 and p50/p50 bands and the ratio displayed in the panel at the right. (E) Supershift EMSA analysis of the 1h lysates from (D) using either no antibody (−) or antibodies to p50 or p65. (F) EMSA for NF-κB and control CREB binding following 24 h or CD3/CD28 stimulation.