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. 2020 Jun 19;11:1087. doi: 10.3389/fimmu.2020.01087

Figure 3.

Figure 3

MDM2 stabilizes the protein level of human FOXP3. (A) MDM2 knockdown assay was performed in human iTreg cells using MDM2 shRNA-carrying lentiviruses, and the mRNA expression levels of MDM2 and FOXP3 were examined by qRT-PCR (n = 3). Error bars represent mean ± S.D. ***p < 0.001; ns, not significant. (B,C) Myc-tagged FOXP3 and doses of Flag-tagged MDM2 were co-expressed in HEK293T cells, and Myc-FOXP3 expression was detected 48 h post-transfection by Western blot. (D,E) Myc-tagged FOXP3 and doses of Flag-tagged wild-type (WT) MDM2 or C478S MDM2 were co-transfected into HEK293T cells, followed by Western blot analysis for Myc-FOXP3 expression after 48 h. (F,G) HEK293T cells were co-transfected with Myc-tagged FOXP3 and Flag vector, Flag-tagged WT MDM2, or C478S MDM2, and were treated with the protein synthesis inhibitor cycloheximide (CHX) for 0, 4, 8, or 12 h before harvesting, followed by the detection of Myc-FOXP3 half-life. The above data are derived from more than three independent experiments.