Foxp3 acetylation prevents proteasomal degradation. Flag-Foxp3–transfected HEK 293 cells were treated with 2μM MG132 (A), 100nM epoxomycin, or 10μM lactacystin (B) for 16 hours to inhibit proteasome function and/or HDACi TSA 100nM and NAM 2.5mM also for 16 hours. Foxp3 expression was analyzed using a Foxp3 antibody, and equal loading was verified by analyzing tubulin expression. (C) Cells were transfected with equal amounts of Flag-Foxp3. Half of the cells were treated with or without TSA 100nM and NAD 2.5mM for 16 hours (right panel) and 5 μg/mL CHX for the indicated time points. Foxp3 expression was analyzed using a Flag antibody, and tubulin expression was used as a loading control. Results are representative of at least 3 independent experiments. (D) Flag-Foxp3 or a Flag-tagged Foxp3 mutant in which all the lysines are mutated to arginines (Flag-Foxp3 K22xR) was transfected into HEK 293 cells. The cells were treated with the HDACi TSA (100nM) and NAM (2.5mM) for 16 hours. Cell lysates were made and immunoblotted for Flag and tubulin as control. (E) HEK 293 cells were transfected with Flag-Foxp3, treated with or without TSA 100nM and NAD 2.5mM for 16 hours, and cell lysates were immunoprecipitated using anti-Flag beads and analyzed using anti-ubiquitin and anti-Flag as transfection control.