Figure 6. Phosphorylation of Nur77 promotes 14-3-3 binding.

(A) FLAG–Nur77 was transfected into HEK-293 cells, which were then serum starved for 16 h. The cells were then left unstimulated or stimulated with 400 ng/ml PMA for 60 min. FLAG–Nur77 was immunoprecipitated from 0.5 mg of lysate and immunoprecipitates were blotted for total and phospho-Ser³⁵⁴ Nur77. 14-3-3 overlays were performed as described in the Materials and methods section. (B) HEK-293 cells were transfected with FLAG–Nur77 and/or GST–14-3-3 as indicated. After transfection, cells were serum starved for 16 h and either treated with 400 ng/ml PMA for 40 min or left unstimulated. Cells were lysed and Nur77 immunoprecipitated using anti-FLAG antibodies or 14-3-3 pulled down using glutathione–Sepharose as indicated, and precipitates were immunoblotted for Nur77 or GST. Total cell lysates was immunoblotted for phospho-Ser³⁵⁴ Nur77. (C) FLAG–Nur77 was transfected into HEK-293 cells, which were then serum starved for 16 h. The cells were then left unstimulated or stimulated with 400 ng/ml PMA for 60 min. FLAG–Nur77 was immunoprecipitated from 0.5 mg of lysate and immunoprecipitates were blotted for either total Nur77 or 14-3-3 (using a pan 14-3-3). Cell lysates were also blotted for phospho-Ser³⁵⁴ Nur77 and total 14-3-3. (D) HA–PFK was transfected into HEK-293 cells, which were then starved for 16 h and then stimulated with IGF (10 ng/ml) for 15 min. HA–PFK2 was immunoprecipitated using an HA antibody and the immunoprecipitates were blotted using a pan 14-3-3 or HA antibody. Cell lysates were also blotted for phospho-PKB and total 14-3-3.