Fig. 4. CARMA1-dependent IKKβ activity contributes to the nuclear localization of c-Maf.

(A) Jurkat cells stably expressing c-Maf were transfected with IKKα- or IKKβ-specific shRNAs and then were selected with puromycin. Nuclear extracts were prepared from the indicated stable cell lines, and the DNA binding activity of c-Maf was analyzed by EMSA with ³²P-labeled probes containing c-Maf– or Oct-1–binding sites. Data are representative of three independent experiments. (B to D) CARMA1-deficient Jurkat cells expressing c-Maf were stably transfected with plasmid encoding constitutively active IKKβ (IKKβ-SSEE) and then were subjected to (B and C) EMSA (nuclear extracts) or (D) Western blotting analysis (cytoplasmic extracts) with antibodies against the indicated proteins. In (C), nuclear extracts from the stimulated cells were treated with SAP before the gel-shift analysis was performed. Blots are representative of two or three experiments. (E) CD4⁺ splenocytes from IKKβ^F/F mice were stimulated with plate-bound anti-CD3 and anti-CD28 antibodies in the presence of IL-4 for 5 days. On days 5 and 6, the cells were infected with RV-GFP-Cre virus (+cre) or RV-GFP control virus (control). GFP⁺ cells were sorted on day 7, rested overnight in complete medium, and restimulated with antibodies against CD3 and CD28 for 2 hours. Nuclear and cytoplasmic fractions were prepared and subjected to Western blotting analysis with antibodies against the indicated proteins. Blots are representative of two experiments. (F) CD4⁺ splenocytes from the indicated mice were stimulated with plate-bound antibodies against CD3 and CD28 in the presence of IL-2 for 48 hours. Nuclear and cytoplasmic extracts were prepared and analyzed by Western blotting with antibodies against the indicated proteins. The experiment was repeated twice with similar results.