Fig. 1.

FOXP3 is acetylated, which is promoted by TIP60. (A) TIP60 expression in both human CD4⁺CD25⁺ T cells and CD4⁺CD25⁻ T cells. FOXP3 and β-actin expression levels were also analyzed by immunoblotting with 221D and anti-β-actin antibodies. (B) Nuclear colocalization of endogenous FOXP3 with TIP60 in CD4⁺CD25⁺ T cells. Human CD4⁺CD25⁺ T cells were stimulated for 2 h with PMA/ionomycin, fixed, permeabilized, and stained by anti-FOXP3 hFOXY (eBioscience), in conjunction with rabbit anti-TIP60 (Upstate) as indicated. Cell nucleus was demonstrated by DAPI staining (blue panel). (C) FOXP3 associates with TIP60 in vivo. HEK 293T cells were cotransfected with expression plasmids for FLAG-TIP60, or HA-FOXP3a as indicated, immunoprecipitated with anti-FLAG M2, followed by Western blotting with anti-FOXP3 221D, or anti-FLAG M2. (D and E) TIP60 promotes FOXP3 acetylation. (D) HEK 293T cells were cotransfected with HA-FOXP3a and an increasing amount of FLAG-TIP60 as indicated, then immunoprecipitated either with acetylated-lysine Ac-K-103 (Upper) or with anti-HA F-7 probe (Lower), followed by Western blotting with HRP-HA. (E) HEK 293T cells were cotransfected with HA-FOXP3a and FLAG-TIP60 as indicated, then immunoprecipitated with anti-HA, followed by Western blotting either acetylated-lysine (Cell Signaling no. 9441; Upper) or HA-HRP (Lower). (F) FOXP3 is acetylated in human CD4⁺CD25⁺ T cells. Nuclear extracts from Jurkat E6.1 T cells and human CD4⁺CD25⁺ T cells were immunoprecipitated with anti-FOXP3 hFOXY, or control IgG, then analyzed with rabbit anti-acetyl-lysine (Upstate) (Upper) and reprobed with anti-FOXP3 221D (Lower).