c-Jun phosphorylation is required for its interaction with FoxP3. (A) Xpress-tagged c-Jun expression plasmids were cotransfected without or with Myc-tagged FoxP3 into HEK 293 cells. Cells were treated with JNK inhibitor SP600125 at the indicated doses for 6 hours and then collected. FoxP3 protein was immunoprecipited with anti-Myc antibody, and the interaction of c-Jun was detected with anti-Xpress antibody (top panel). The same membrane was reprobed with anti-FoxP3 antibody (second panel from top). The protein levels of c-Jun, phosphorylated c-Jun, and actin in the whole cell lysates were detected by Western blotting (bottom 3 panels). (B) c-Jun and FoxP3 expression plasmids were cotransfected into HEK 293 cells and cultured for 36 hours. Transfected cells were then treated with or without 50 nM of JNK inhibitor SP600125 for 6 hours. The protein of FoxP3 in the lysates of transfected cells was immunoprecipitated with anti-Myc antibody. The immunoprecipitates were analyzed by SDS-PAGE and then detected with anti-phospho–c-Jun antibody (top panel). (C,D) Xpress-tagged c-Jun or its mutants, including mutation of the 63, 73 serine to alanine (S2A), deletion of the δdomain (Δδ), and mutation of the 63, 73 serine and 91, 94 threonine to alanine (S2A/T2A), were cotransfected without or with Myc-tagged FoxP3 into 293 cells. FoxP3 protein in the lysates from transfected cells was immunoprecipitated with anti-Myc antibody; the bound c-Jun or its mutants were detected with anti-Xpress antibody (top panels). The same membranes were reprobed with anti-FoxP3 antibody (second panel from top). The expression of c-Jun or its mutants and actin in the whole cell lysates was determined with Western blotting as controls (bottom 2 panels).