CPDT and SF block Nrf2 ubiquitination.

NBT-II cells were co-transfected for 24 h with expression plasmids for Nrf2 (pEF/Nrf2), Keap1 (pcDNA1/Keap1) or ubiquitin (pMT107-His-Ub, a polyhistidine-tagged ubiquitin expression plasmid), followed by treatment for 6 h with MG132 (25 µM), MG132 (25 µM) plus CPDT (50 µM), or MG132 (25 µM) plus SF (8 µM), from which cytoplasmic and nuclear samples were prepared and analyzed by IB of various proteins. For detection of ubiquitinated Nrf2, the samples were prepared under denatured conditions and then subjected to IP with anti-Nrf2, followed by IB with an anti-His-HRP-conjugated antibody (for detection of ubiquitinated Nrf2). Equal amounts of nuclear and cytoplasmic proteins were used. The arrow points to the Ub band.