HEK 293 cells were transfected with an expression plasmid for FLAG-Nur77. Cells were then starved for 16 hours and incubated with H89, Ro 318220, wortmannin, SB 203580, U0126 or PD 184352 at the concentrations indicated. Cells were then stimulated with either 400 ng/ml PMA for 60 min or 100 ng/ml EGF for 10 min. Cells were then lysed and FLAG-Nur77 was immunoprecipitated using an antibody against the FLAG tag. Immunoprecipitates were subjected to SDS-Page analysis before being transferred to nitrocellulose membrane and blotted with the appropriate antibody. The upper panels were probed with the anti-Nur77 phospho Ser 354 at 1 μg/ml in the presence of 10 μg/ml non-phosphorylated peptide, while the lower panels were probed with anti-Nur77 (18- 32). Binding of the primary antibody was detected using rabbit peroxidase conjugated anti- sheep IgG (1 in 10, 000 dilution, Pierce) followed by enhanced chemiluminscence (ECL, Amersham).