p300 promotes Foxp3 acetylation. (A) Flag immunoprecipitation from lysates from Flag-Foxp3–transfected cells. HEK 293 cells were treated with HDACi: + indicates 50 nm TSA and 1mM NAM; or ++, 250 nm TSA and 5mM NAM for 16 hours. Equal amounts of immunoprecipitated Foxp3 were separated by SDS-PAGE, and Western blots were probed for acetylated lysines (Ac-Lys) or Flag. (B) Cells were transfected with Flag-Foxp3 and/or HA-p300. Flag-Foxp3 was immunoprecipitated with anti-Flag beads and analyzed using acetyl lysines (Ac-Lys), Flag, or HA antibodies. (C) Cells were cotransfected with Flag-Foxp3 and HA-p300 or HA-TIP60 and immunoprecipitated using anti-Flag beads. Immunoblots were analyzed with acetyl-lysine (Ac-Lys) antibody, anti-Flag, or anti-HA. Cells were cotransfected with Flag-Foxp3, HA-Tip60 (D), or HA-p300 (E). Cell lysates were coimmunoprecipitated using anti-HA beads and analyzed using anti-Flag or anti-HA. (F) GST-Foxp3 fusion protein coupled to Sepharose beads was incubated with [14C]-labeled acetyl-CoA and p300 or the acetylase dead p300mutAT2; samples were separated on SDS-PAGE and analyzed using films sensitive for radioactivity. Results are representative of at least 3 independent experiments. *Aspecific band. IP indicates immunoprecipitation; and WB, Western blot.