Acetylation modulates Foxp3 protein levels. (A) Representative examples of cells that were transfected with only Foxp3 (green; top panel). Subcellular distribution of cells that were cotransfected with Foxp3 (green) and p300 (red) are shown in the bottom panel. p300 was localized using an anti-p300 antibody that recognizes both endogenous and ectopically expressed p300. Colocalization of Foxp3 and p300 is indicated in yellow. (B) Foxp3 function was assessed by evaluating IL-2 promoter reporter activity. IL-2 promoter luciferase activity was analyzed in HEK 293 cells by cotransfecting NFAT with Foxp3 del E250 (▭), Foxp3 (), or Foxp3 with p300 (). Repression of IL-2 luciferase activity by wild-type Foxp3 was set as 100%. Values were all normalized for cotransfected Renilla. **P < .01. (C) HEK 293 cells were transfected with Flag-Foxp3 and treated with HDACi: + indicates 50 nm TSA and 1mM NAM; ++, 250 nm TSA and 5mM NAM. Immunoblots were probed for Flag expression or tubulin as loading control. (D) Cells were transfected with Flag-Foxp3 and increasing amounts of HA-p300. Western blots were incubated with antibodies against Flag, HA, or tubulin as indicated. (E) HEK 293 cells were transfected with Flag-Foxp3 or Flag-Foxo3 with or without HA-p300. Cells were treated with 100nM TSA and 2.5mM NAM for 16 hours (HDACi). Data are representative of at least 3 independent experiments.