NR4A1 transcriptional activity in cortical neurons depends on its nuclear localization, phosphorylation, and turnover. A, NR4A1 activity expressed as a percentage of untreated controls impeded after stimulation with 25 ng/ml BDNF for 3 h (mean ± SEM of four independent experiments; unpaired t test, *p = 0.042). NurRE-Luc reporter was cotransfected with recombinant NR4A1 in primary cortical neurons by electroporation and Luciferase activity monitored in lysates. B, Antibodies to detect the nucleo-cytoplasmic isoforms (pan-NR4A1), cytosolic isoform (cyto-NR4A1), and phosphorylated isoforms (p-NR4A1 at Ser350) of recombinant NR4A1 electroporated in primary cortical neurons. Ratio of proteins in cytosol (C) vs nuclear (N; means of four independent samples/group) altered after stimulation with 25 ng/ml BDNF for 3 h. HSP90 and HDAC2 are loading controls. C, NurRE-Luc reporter activity in primary cortical neurons transfected with the indicated constructs. Data normalized to GFP controls (mean ± SEM of three independent experiments; ANOVA post hoc Dunnett's test comparing WT and mutants, *p < 0.0005). D, Deletion of Ser350 or NLS sequences altered NR4A1 C/N ratio in primary cortical neurons (means of four independent samples/group normalized to WT+BDNF). E, Polyubiquitination of NR4A1 immunoprecipitated from lysates of primary cortical neurons stimulated with 25 ng/ml BDNF for 3 h. F, Withdrawal of doxycycline for the indicated time destabilized NR4A1 recombinant protein levels in primary cortical neurons. Mean ± SEM of four independent experiments; ANOVA post hoc Sidak's test for interaction of BDNF and time, *p < 0.0001.