Fig. 1. K¹¹⁰ and K⁵²⁵ are SUMO-acceptor lysines in mouse Nrf2. Wild-type and mutant K110R, K525R, 2K (double mutant; K110R/K525R), GST-mNrf2 fusion proteins were purified from E. coli as described under Experimental Procedures. After purification, purified protein was resolved on 7.5% SDS-PAGE. A. Comassie brilliant blue stained gel picture. The purified protein migrates at ~130 kDa marked by *. The identity of the purified protein was validated further with immunoblot analysis. B. Immunoblot analysis of purified protein from panel A with anti-Nrf2 antibody. C. Immunoblot analysis of purified protein from panel A with anti-GST antibody. In vitro sumoylation assay was performed with kits (Active Motif, Carlsbad, CA), using 0.5 mg of the purified fusion proteins as substrates and products of the reaction were separated on 7.5% SDS-PAGE and analyzed by western blotting using the indicated antibodies. The lower panel is showing the bottom part of the same Western blot reacting with anti-GST antibody showing GST protein (~32 kDa). D. SUMO-2 conjugated GST-mNrf2 were detected with anti-SUMO-2/3 antibody (upper panel) and detected with anti-Nrf2 antibody (lower panel). E. Densitometric analysis of the immunoblots in panel D was done and normalized values were plotted to find relative SUMOylation level with respect to total Nrf2. Values plotted (E) (K110R 7%, K525R 20% and 2K 16%) are the ratio of SUMO-2 conjugated mNrf2 to total mNrf2. Results are mean ±S.E. (n=3). Statistical analysis was performed with one-way analysis of variance with Dunnett's multiple comparison test. ****, statistically different (p<0.001). The data in B-E are representative of results from three separate experiments.