Nurr1 is SUMOylated by SUMO-2.
(A) Schematic representation of Nurr1 (bottom) showing the position of four putative SUMOylation sites according with SUMOplotTM software analysis (middle). The table shows the sequences of the potential SUMOylation sites of rat Nurr1 sorted from the highest score. Putative SUMO acceptor lysines (K) are highlighted, and potential SUMO sites are underlined. (B) COS-7 cells were transfected with plasmids expressing HA-Nurr1, Ubc9, and SUMO-1, SUMO-2 or SUMO-3. Cells were harvested 48 hours post-transfection and lysed directly in loading buffer containing the SUMO-isopeptidase inhibitor N-ethylmaleimide 20 mM, and fractionated in SDS-PAGE. Representative western-blot with anti-HA (upper) and anti-SUMO-2 (bottom) antibodies. (C) Quantitative densitometry analysis of Nurr1-SUMO-2 signal described in (B), using Image J software. Data correspond to the mean ± S.E.M. of 3 independent experiments for each condition. Statistical significance was estimated by the non-parametric Mann-Whitney U-test. * p<0.05 (Nurr1+Ubc9+SUMO-2 v/s Nurr1). (D) Total lysates from COS-7 cells transfected with HA-Nurr1, Ubc9 and SUMO-2 were immunoprecipitated with an anti-Nurr1 antibody or control IgG. The immunoprecipitates were analyzed in western blots with anti-HA antibody. Bands for immunoglobulin are indicated as IgG heavy chain (H).