Figure 5.

TCR/CD28 signaling up-regulates RNF31 expression in Treg cells. A, male C57BL/6 mice were sacrificed, lymph nodes were isolated, followed by the detection of RNF31 and Foxp3 in naive CD4⁺Foxp3⁺CD44⁻ Treg cells and effector/memory CD4⁺Foxp3⁺CD44⁺ Treg cells (n = 4). **, p < 0.01. B–D, in vitro expanded human Treg cells were rested (cells were removed of anti-CD3/CD28 beads, and 100 units of IL-2 was added for sustaining survival) for 2 days and were then re-stimulated with anti-CD3/CD28 Ab (1 μg/ml) plus 500 units of IL-2 as indicated. Cells were harvested, followed by immunoblot analysis of endogenous FOXP3 and RNF31. E, rested in vitro expanded Tregs were challenged with TCR/CD28 activation as indicated, followed by immunoblot analysis for detection of endogenous RNF31, FOXP3 expression, and ubiquitination of FOXP3. The arrow in D and E indicates monoubiquitinated FOXP3.