The activation of ERK2 and JNK1 is reduced in anergic iTregs induced by coculture with IL-10–modulated DCs. CD4⁺ T cells were cocultured with allogeneic IL-10DCs or mDCs for 5 days to induce iTregs (R) and Teffs (E). Subsequently, T cells were restimulated by anti-CD3 mAb. Lysates of T-cell populations were prepared at 0, 15, 60, and 240 minutes after restimulation. ERK2 and JNK1 were immunoprecipitated with anti-ERK2 and anti-JNK1 antisera. MBP and GST–c-Jun served as specific substrates for ERK2 (panel A; top) and JNK1 (panel B; top) in in vitro kinase assays to analyze their activation. Western blots using antibodies against ERK2 and JNK1, respectively, demonstrated equivalent protein loading of both T-cell populations (panels A-B; bottom). One representative of 3 independent experiments with similar results is shown.