Fig. 5. SUMO-acceptor lysines are needed for stability of Nrf2. HEK293T cells were transfected with either wild-type Flag-hNrf2 (WT), or mutant FLAG-hNrf2: K110R, K533R, or 2K (double mutant, K110R/K533R). Twenty-four hours after transfection the cells were treated with 100 µM cycloheximide (CHX) for 0, 5, 15, 30 and 60 min. Total cell lysate were prepared and separated on a 7.5% SDS-PAGE and analyzed by Western blotting using anti-Flag and anti-β-Actin antibodies. A, Representative blots are shown. B. Densitometric quantitation of the blots in A. Values for Flag-Nrf2 were normalized with actin and normalized percent values plotted to find the half-life of mutant Flag-hNrf2 with respect to wild type. UN-SCAN-IT software (Silk Scientific, Inc., Orem, UT) was used for quantitation. The values plotted are means ± S.E (n=3).