FIGURE 4.

Foxp3 lacking CDK consensus elements has increased protein stability. A, CD4⁺25⁻ T lymphocytes were transduced with vector control, wild type Foxp3, or the S/T→A-Foxp3 mutant. LTR, long terminal repeat; IRES, internal ribosomal entry site. B, transduced cells were rested overnight and restimulated for 4 h with plate-bound CD3 and CD28 antibodies, incubated with 25 μg/ml CHX, and harvested at the indicated times. Whole cell extracts from treated cells were analyzed by SDS-PAGE, blotted to membranes, and probed with Foxp3 antibody. One representative image of three experiments is shown. C, average stability of wild type and S/T→A-Foxp3 over three experiments is shown. Half-life was calculated based on these values. Error bars are S.E. for biological replicate cultures. D, Foxp3 transcription normalized to actin is shown. Message level is represented in arbitrary units, before the addition of CHX to transduced cells.