HEK 293 cells were transfected with an expression plasmid for FLAG-Nurr1. Cells were then starved for 16 hours and stimulated with either 400 ng/ml PMA (left handside panel) for 60 min or 100 ng/ml EGF (right handside panel) for 10 min. Cells were then lysed and 30 μg of soluble protein lysate was subjected to SDS-Page analysis before being transferred to nitrocellulose membrane and blotted with the appropriate antibody. The upper panels were probed with anti-Nur77 phospho Ser 354 (which recognises Nurr1 phosphorylated on Ser 347) at 1 μg/ml in the presence of 10 μg/ml non-phosphorylated peptide, while the lower panels were probed with anti-Nurr1 (27-41) at 1 μg/ml. 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).