(A) Detection of Itch in Th1 and Th2 cells upon ionomycin (Iono) treatment. Differentiated Th1 or Th2 cells were left unstimulated or stimulated with ionomycin (50 ng/ml) for 16 hours. Cell lysates were probed with anti-Itch antibody. The intensity of the protein bands was quantitated, and the relative amount of Itch in untreated WT T cells was given an arbitrary value of 1.0. The same membrane was reprobed with anti-actin to assess equal loading of the samples. (B) Th2 cells were first treated with different concentrations of ionomycin for 16 hours, and the cell lysates were immunoblotted with antibodies as indicated. (C) Th2 cells were left untreated or treated with ionomycin, washed, and then stimulated with anti-CD3 plus anti-CD28. The nuclear fractions were isolated and immunoblotted with anti-JunB, –c-Jun, or –histone H1 antibody. (D) Th2 cells left untreated or treated with ionomycin were measured for the mRNA levels of c-Jun, JunB, or JunD by real-time PCR. (E) T cells from PBS-treated or OVA-tolerized WT or Itch^–/– OTII mice were analyzed for the protein levels of the indicated molecules by immunoblotting. (F) Lysates from cells treated similarly to those described above were immunoblotted with the indicated antibodies. (G) T cells as in E were restimulated with OVA peptide plus irradiated splenocytes. The nuclear fractions were isolated and immunoblotted with the indicated antibodies.