Fig. 2. SUMO-2 acceptor K¹¹⁰ and K⁵³³ are needed for efficient transcriptional activity of Nrf2. A, Schematic of the HO-1-ARE-Luc (firefly luciferase, experimental) and Tk-Luc (control) promoters used in the luciferase reporter assay. HEK293T cells were transfected with 100 hM control siRNA or Nrf2 siRNA for 48 hrs. B. Quantitative RT-PCR analysis of the levels of Nrf2 mRNA in HEK293T cells after knockdown with siC: scrambled control siRNA, siN: siRNA against 3'UTR of endogenous Nrf2. Results are mean ±S.E. (n=3). ** indicate there is a statistically significant difference. C. Western blot analysis of Nrf2 protein levels after siRNA mediated knockdown (* denotes the non-specific reactivity of the antibody). D. To monitor Nrf2 mediated transcriptional activation shown is the relative luciferase activity driven by HO1-ARE promoter in HEK293T cells treated with either control siRNA (siC) or siRNA against Nrf2 (siN) without activation, or with siRNA and activation by treatment with As2O3 (siC_As2O3 and siN_As2O3). Results are mean ±S.E. (n=9). ** indicates there is a statistically significant difference in all observations compared to siC. E. To study the significance of the SUMO-acceptor lysine in Nrf2 rescue experiments were performed using plasmid expressing either wild type Flag-hNrf2 (WT) or different mutant Flag-hNrf2 (K110R, K533R or 2K, K110R/K533R double mutant) in the HEK293T cells treated with siRNA against Nrf2. pCMV-Flag vector (pFlag) alone was used as a control. Shown is the relative luciferase activity driven by HO1-ARE promoter mediated by Flag-tagged hNrf2 wild type or mutants. Results are mean ±S.E. (n=9). ****, statistically different (p<0.001), indicates there is a statistically significant difference in all observations compared to empty vector (pFlag) transfected cells. F. immunoblot showing the expression of recombinant Flag-tagged Nrf2 wild type (WT) and mutant (K110R, K533R AND 2K) proteins after transient transfection in HEK293T cells using Flag antibody. Empty Flag-vector (pFlag) is used as a negative control. β-Actin is used as a loading control.