(A) WT or MEKK1ΔKD mice were tolerized with soluble OVA or treated with PBS as control, plus simultaneous immunization with OVA in alum. T cells were stimulated for proliferation in vitro by OVA antigen peptide plus APCs. (B) The production of IL-4 from the in vitro–cultured cells was measured. (C and D) WT or JNK1^–/– mice were subjected to tolerance induction. The cell proliferation and IL-4 production were determined. (E) Adoptive transfer of PBS-treated or OVA-tolerized T cells from WT, MEKK1ΔKD, or JNK1^–/– mice. The purified CD4⁺ T cells were labeled with CFSE, and the recipient mice were immunized with OVA in alum. The intracellular IL-4 production and the CFSE profile of gated Thy1.2⁺ T cells were examined. (F and G) Lysates from T cells from PBS-treated or OVA-tolerized WT, MEKK1ΔKD, or JNK1^–/– mice were immunoblotted with antibodies as indicated. (H) T cells from OVA-tolerized WT mice were subjected to immunoprecipitation with anti-Itch (top panel) or anti-JNK1 (bottom panel), with anti-p65 of NF-κB as a control. The immunoprecipitates were blotted with the indicated antibodies. (I) The interaction of JNK1 and Itch was analyzed from cells without or with ionomycin treatment. (J) T cells were left untreated or tolerized with ionomycin, and the cells were restimulated with anti-CD3 plus anti-CD28 for 10 minutes. Lysates were immunoprecipitated with anti-JNK1 antibody and immunoblotted with anti–phospho-JNK1 (p-JNK1).