Figure 7  Foxp3 Antagonizes CREB Activation by Blocking Recruitment of Coactivator Protein p300
(A and B) HEK 293T cells (5 × 105) were seeded into six-well plates 1 d prior to transfection with a CREB or an HTLV-I LTR luciferase reporter vector (200 ng) in the presence or absence of a Foxp3, DFKH, or control expression vector (500 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng). Forskolin (10 μM) was added to the appropriate reactions 20 h posttransfection. Reactions were assayed 24 h posttransfection for luciferase activity (A) and CREB-1 and ATF-2 DNA-binding activity (B).
(C) HEK 293T cells (5 × 105) were seeded into six-well plates 1 d prior to transfection with a Gal4 luciferase reporter vector (200 ng) in the presence or absence of Gal4-BD-CREB-1 (500 ng) along with a control, Foxp3, or p300 expression vector (500 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng). Cells were harvested at 24 h posttransfection and analyzed for luciferase activity. Relative luciferase activity in (A and C) was normalized to the internal reference control.
(D) Whole-cell extracts from HEK 293T cells transfected with a control (EGFP) or Foxp3 expression vector together with a p300-HA expression vector were immunoprecipitated with anti-HA monoclonal antibody. Proteins were then separated by SDS-PAGE and immunoblotted with anti-Foxp3 (ab10563) to detect immunoprecipitates and with anti-HA for the lysates.