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 TNFα-promoter, as determined by ChIP assay (Fig. 3A). To further confirm GSK3β involvement, we performed TNFα-luciferase reporter assays in RAW264.7 cells cotransfected with a kinase-dead mutant of GSK3β (GSK3β-K85R mutant, GSK3β-KD). GSK3β-KD expression abolished the Nurr1-mediated transrepression of the TNFα-promoter in a dose-dependent manner (Fig. S7B). Furthermore, knockdown of GSK3β completely prevented Nurr1-mediated iNOS repression (Fig. 3H). We further validated the contribution of phospho-S468 in p65 by exchanging S468 for alanine (S468A). The p65 S468A mutant, but not wild-type p65, reversed Nurr1-mediated iNOS repression in RAW264.7 cells in a dose-dependent manner (Fig. 3I and Fig. S7E). Finally, GSK3β stimulated the in vitro interaction of Nurr1 with wild-type p65 but not with p65-S486A (Fig. S7C).
