Fig. 4. CoREST repressor complex is required for Nurr1-mediated repression.

A. Corepressor requirements in Nurr1-mediated repression. iNOS-luciferase and Nurr1-expression or control vector as well as siRNAs against the indicated corepressors were transfected into RAW264.7 cells and iNOS-promoter activity was assayed. *, p<0.01 compared to Nurr1 with control siRNA. B. Interaction of Nurr1 and CoREST in BV2 cells. Co-IP was performed with anti-CoREST antibody and Western blots were developed with anti-Nurr1 antibody. C. Effect of siRNAs against the indicated targets on Nurr1-mediated repression of iNOS-promoter activity. *, p<0.01, **, p<0.001 compared to Nurr1 with control siRNA. D. NLK in vitro kinase assay using GST-Nurr1 and GST-CoREST as substrates. Arrows indicate phosphorylated GST-Nurr1 and autophosphorylation of NLK. The migration position of GST-CoREST is indicated by an asterisk. GST substrates and methods are provided in Supplemental Experimental Procedures and Figure S7D. E. Effect of NLK knockdown on interaction of Nurr1 and CoREST. BV2 cells were transfected with siRNA against NLK or control siRNA. Co-IP of Nurr1 and CoREST was performed as described in B. F. Recruitment of Nurr1, CoREST and p65 to the iNOS promoter in BV2 cells shown by ChIP assay. Data represent fold enrichment of iNOS-promoter precipitated by the indicated antibodies compared to control IgG as determined by qPCR. G. ChIP analysis of Nurr1 on the iNOS promoter in the SN before and after LPS stimulation. Data are shown as averages of fold enrichment against control IgG and SD. H. Effect of Nurr1 knockdown on recruitment of CoREST (left) and p65 (right) to the TNFα promoter in BV2 cells. ChIP data are shown as fold enrichment over control IgG.