TRAF6 and IRAK1 are bona fide targets of miR-146a in mouse T cells and are possible mediators for its regulation of NF-κB activity. (A and B) TaqMan Q-PCR analysis of miR-146a expression (A; shown as mean of duplicates ± SEM) and Western blot analysis of TRAF6 and IRAK1 expression (B) in WT or miR-146a^−/− SP/LN cells stimulated with soluble anti-CD3 + anti-CD28 (1 µg/ml each) for 3 d. Data are representative of three independent experiments. (C and D) TaqMan Q-PCR analysis of miR-146a expression (C; shown as mean of duplicates ± SEM) and Western blot analysis of TRAF6 and IRAK1 expression (D) in WT SP/LN cells transduced with MIG–miR-146a or control MIG retroviral vectors and stimulated with soluble anti-CD3 + anti-CD28 (1 µg/ml each) for 3 d. Data are representative of three independent experiments. (E) Western blot analysis of TRAF6 and IRAK1 expression in activated WT or miR-146a^−/− CD4 and CD8 T cells. CD4 and CD8 T cells were purified through MACS sorting from WT or miR-146a^−/− SP/LN cells stimulated with soluble anti-CD3 + anti-CD28 (1 µg/ml each) for 3 d. Data are representative of three independent experiments. (F–J) WT SP/LN cells were stimulated with soluble anti-CD3 + anti-CD28 (1 µg/ml each) and transduced with MIG-TRAF6, MIG-IRAK1, or control MIG retroviral vectors. By day 3, >90% of total cell culture was activated T cells (sum of CD4⁺ and CD8⁺ cells). Data are representative of at least three independent experiments. *, P < 0.05; **, P < 0.01 (MIG-TRAF6– or MIG-IRAK1–transduced samples in comparison with the corresponding MIG-transduced controls). (F) Western blot analysis of TRAF6 and IRAK1 expression in day 3 transduced cells. (G) EMSA analysis of NF-κB activity of 10 µg of nuclear extracts from day 3 transduced cells rested and restimulated with plate-bound anti-CD3 (10 µg/ml) + anti-CD28 (1 µg/ml) for 30 min. Representative EMSA image and the measurements of NF-κB in arbitrary units (mean ± SEM; cells transduced with MIG = 1) from three independent experiments are shown. (H) Relative CD4 and CD8 T cell number (gated as CD4⁺ and CD8⁺, respectively, cell count of MIG-transduced T cells = 1) on day 3. Data are presented as mean of duplicate culture ± SEM. (I) Annexin V staining analysis of apoptosis of transduced CD4 and CD8 T cells (gated as CD4⁺GFP⁺ and CD8⁺GFP⁺, respectively) on day 4. Data are presented as mean of duplicate culture ± SEM. (J) ELISA analysis of effector cytokine production from transduced cells on day 3. Data are presented as mean of duplicate culture ± SEM. (K–N) WT or miR-146a^−/− SP/LN cells were stimulated with soluble anti-CD3 + anti-CD28 (1 µg/ml each) and transfected with nonsilencing control siRNA (siCtrl) or siRNA specific for mouse TRAF6 (siTRAF6) or IRAK1 (siIRAK1). Representative data from two independent experiments are shown. *, P < 0.05; **, P < 0.01 (miR-146a^−/− cells transduced with various siRNAs in comparison with WT cells transduced with control siRNA). (K) Western blot analysis of TRAF6 and IRAK1 expression in the siRNA-transfected cells on day 3.5. (L) [³H]Thymidine incorporation analysis of proliferation of the siRNA-transfected cells on day 3. Data are presented as mean of duplicate culture ± SEM. (M) Annexin V staining analysis of apoptosis of the siRNA-transfected CD4 and CD8 T cells (gated as CD4⁺ and CD8⁺, respectively) on day 3.5. Data are presented as mean of duplicate culture ± SEM. (N) ELISA analysis of effector cytokine production from the siRNA-transfected cells on day 3.5. Data are presented as mean of duplicate culture ± SEM. Numbers under Western blots denote relative amounts normalized to β-actin or PLC-γ1 expression for each sample.