Figure 7. Activation of NBRE and NurRE reporter plasmids.

(A) HEK-293 cells were transfected with a Renilla luciferase (to control for transfection efficiency) and NBRE or NurRE luciferase reporters along with either empty vector or expression vectors for wild-type (WT), S354A or S354D or an empty vector (EV) as indicated. Cells were lysed and luciferase activity was measured and normalized for the Renilla control. Error bars represent the S.E.M. for 16 (NBRE) or 20 (NurRE) replicates. (B) Experiments were performed as described in (A) except that cells were serum starved for 16 h and then stimulated with PMA (400 ng/ml) for 0–8 h as indicated. Black bars represent wild-type Nur77 and white bars represent S354A Nur77. Error bars represent the S.E.M. for 16 (NBRE) or 20 (NurRE) replicates. (C) Anti-FLAG immunoblot showing the relative expression levels of the Nur77 wild-type and Ser³⁵⁴ mutant constructs in HEK-293 cells.