FIGURE 9.

SUMO-1 sumoylation of Nrf2 and MafG at specific sites affect their interaction and GCLC expression but not Nrf2/c-Myc interaction or Nrf2 ubiquitination. A, effect of mutating the SUMO-1 sites on Nrf2 and MafG (see supplemental Fig. 1) on Nrf2/MafG interaction in HSCs. Sumoylation site mutants of Nrf2 and MafG were overexpressed in BSC cells along with their corresponding normal constructs. Nuclear extracts of these cells were immunoprecipitated (IP) with Nrf2 and checked for MafG expression by Western blotting (WB). Results are from two experiments in duplicate. *, p < 0.05; **, p < 0.005 versus empty vector control. B, mutant and wild type constructs were expressed as in A, and the expression of GCLC was examined by Western blotting. Results are from three experiments in duplicate. *, p < 0.05; **, p < 0.005 versus empty vector control. C and D, effect of SUMO-1 knockdown on interaction of Nrf2 with c-Myc protein and ubiquitin. Nuclei from MDI BSC cells were treated with scrambled (SC) or SUMO-1 siRNA, and nuclear extracts were processed for immunoprecipitation of Nrf2 followed by Western blotting for c-Myc (C) or ubiquitin (D). Results are from two experiments in duplicate. E, effect of SUMO-1 knockdown on total ubiquitinated proteins in MDI-treated BSC cells. Total protein extracts from scrambled (SC) or SUMO-1 siRNA cells were subjected to Western blotting using anti-ubiquitin antibody. Results are from two experiments in duplicate.