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. Author manuscript; available in PMC: 2009 Oct 3.
Published in final edited form as: Cell. 2009 Apr 3;137(1):47–59. doi: 10.1016/j.cell.2009.01.038

Fig. 3. Nurr1 acts as an RXR-independent, GSK3.

Fig. 3

β-dependent transrepressor for NF-κB. A. ChIP assay of Nurr1 on the TNFα-promoter in response to LPS and effect of the GSK3β-specific inhibitor SB216763 (SB21). BV2 cells were pre-incubated with DMSO or 30 μM SB21 for 1h followed by LPS stimulation for the indicated times before ChIP assay. Data are displayed as fold enrichment over control IgG. B. Repression activities of Nurr1 mutants. RAW264.7 cells were transfected with wild type Nurr1, P-box mutant (CEAA) and I-box mutant (KLL). iNOS-promoter activity in RAW264.7 cells in response to LPS was measured by luciferase-reporter assay. *, p<0.01 compared to control (Mock). C. Effect of knockdown of Ubc9 on Nurr1 repression of iNOS-promoter activity. *, p<0.01 compared to Nurr1 with control siRNA. D. Identification of the predominant SUMOylation sites of Nurr1. Flag-tag mutants of Nurr1 were transfected into Hela cells. SUMOylation assay was performed as described in Supplemental Experimental Procedure. E. Effect of reconstitution of Nurr1 shRNA-2 BV2 cells with non-targeted (NT) WT and mutant forms of Nurr1 that are not recognized by shNurr1-2. Endogenous iNOS mRNA levels are shown relative to levels in untreated BV2 cells transduced with control shRNA and mock Nurr1 lentivirus. *, p<0.01 compared to mock control cells. F. Effect of LPS stimulation on interaction of Nurr1 and p65 in BV2 cells. Lysates of BV2 cells stimulated with LPS for the indicated times were immunoprecipitated with anti-Nurr1 antibody and Western blots were developed with anti-p65 antibody. G. Effect of SB21 on Nurr1/p65 interaction. BV2 cells were incubated with SB21 at the indicated concentrations for 1h prior to stimulation with LPS. IP and Western blotting were performed as described in F. H. Effect of siRNA against GSK3β on Nurr1-mediated repression of iNOS-promoter activity. Nurr1 expression vector was transfected into RAW264.7 cells together with control siRNA or siRNA against GSK3β *, p<0.01 compared to Nurr1 with control siRNA. I. Effect of S468A mutation of p65 on Nurr1-mediated repression of iNOS-promoter activity.