miR-146 represses TCR-dependent NF-κB signaling in thymocytes by targeting Traf6. (A) Quantitative RT-PCR analysis of miR-146a and miR-146b expression levels in FACS-sorted DP thymocytes, from 5-week-old littermate WT and tPTEN^−/− mice (C57BL/6 background), left unstimulated or stimulated with anti-CD3/CD28 antibodies for 16 hours. Where indicated, DP thymocytes were pretreated with 5 μM or 15 μM LY294002 (LY) for 1 hour before anti-CD3/CD28 stimulation. Expression was normalized to miR-16 (or sno-234 in supplemental Figure 3) and fold change is relative to untreated cells. Flow cytometry analysis shows dose-dependent inhibition of Akt phosphorylation in LY294002-treated cells. (B) Quantitative RT-PCR analysis of Traf6 expression in littermate late premalignant and malignant tPTEN^−/− mice compared with an age-matched WT control (C57BL/6 background). (C) Quantitative RT-PCR analysis of Traf6 expression in 146a1 and 146b transgenic mice compared with littermate controls on WT and tPTEN^−/− backgrounds. (D) Western blot analysis of Stat1 protein expression in total thymocytes and mature T cells from 12-week-old littermate 146a8 transgenic and WT mice. (E) Western blot analysis of Traf6 expression, IκBα degradation, and c-myc induction in TCR-stimulated total thymocytes from 10-week-old 146a1/tPTEN^−/− and 146b/tPTEN^−/− mice and littermate or age-matched tPTEN^−/− controls. Thymocytes were stimulated with PMA and ionomycin for the indicated times. Data in (A-C) are presented as mean ± SD and are representative of 3 independent experiments. Statistics were calculated by Student t test (***P < .001, **P < .01, *P < .05, n.s. not significant).