Since transrepression requires the tethering of NRs to other transcription factors, we tested whether Nurr1 could bind to transcription factors involved in inflammation, such as NF-κB. Co-immunoprecipitation (Co-IP) assays of Nurr1 in BV2 cells showed interaction with NF-κB-p65 that was significantly enhanced by LPS treatment and independent of changes in Nurr1 protein levels (Fig. 3F and Fig. S6A). Phosphorylation of Serine-468 (S468) in p65 is associated with negative regulation of NF-κB signaling (Buss et al., 2004) and can be mediated by GSK3β, which is activated following TLR4 stimulation in human monocytes (Martin et al., 2005). Furthermore, inactivation of GSK3β results in increased NF-κB-dependent transcription of TNFα without changing the kinase activity of the IKK complex or the nuclear translocation of p65 (Buss et al., 2004). Therefore, we hypothesized that S468 phosphorylation of p65 by GSK3β might provide the docking site for tethering of Nurr1. Consistent with this possibility, the GSK3β-specific inhibitor SB216763 (SB21) inhibited the interaction of Nurr1 and p65 in BV2 cells in a dose-dependent manner (Fig. 3G and Fig. S7D) and prevented the recruitment of Nurr1 to the
