Fig. 4. SUMO-acceptor lysines are needed for efficient nuclear localization of Nrf2. HEK293T cells were transfected either with the empty Flag-vector (pFLAG), or Flag-Nrf2 wild-type (WT) or mutant (K110R, K533R) and levels of Flag-tagged Nrf2 were monitored in the cytoplasmic and nuclear enriched factions with or without activation with As₂O₃. A. Immunoblot analysis using anti-Flag antibody on cytoplasmic (C) and nuclear enriched (N) factions for cells without activation. B. with activation by 10 µM As₂O₃ treatment for 4 hrs. Antibody against nuclear protein Fibrillarin was used to validate the enrichment of nuclear proteins in nuclear fraction. C, graphs representing the relative level of nuclear Nrf2 post activation with As₂O₃ compared to WT. Density of both the bands in the panel A and B with anti-Flag antibody were quantitated for Flag-Nrf2 levels in the nuclear enriched fraction, normalized by Fibrillarian. The ration of normalized value of with As₂O₃ over without As₂O₃ are plotted. Results are mean ±S.E. (n=3). *** indicates statistical significance p<0.001 in mutants compared to wild type. C: cytoplasmic fraction, N: nuclear enriched fraction. C: cytoplasmic fraction, N: nuclear enriched fraction. D. immunofluorescence confocal microscopy analysis of Flag-hNrf2 WT or mutant protein with or without activation by As₂O₃ on its nuclear localization. Anti-Flag-Cy3 (red) antibody was used for detection of recombinant Nrf2 WT or mutant, Cell nuclei are painted blue with DAPI. E. Images such as those in D were used to obtain the Pearson coefficients for subcellular colocalization to determine colocalization of Flag-hNrf2 with the nucleus. Normalized values with As2O3 over without As2O3 are plotted. ** indicates statistical significance p<0.001 in mutants compared to wild type.