Arkadia induces Nrf2-dependent ARE-driven gene transcription. A, Schematic of pRK5-HA-Ubiquitin mutant plasmids. Five HA-ubiquitin mutation plasmids were used; HA-ubiquitin harboring a single K→R mutation at position K48 (Ub-K48R) or at position K63 (Ub-K63R), HA-ubiquitin bearing a single Lys at either position 48 (Ub-K48 only) or position 63 only (Ub-K63 only) with the other six Lys residues in ubiquitin mutated to Arg. B, C, HepG2 cells were co-transfected with 0.2 μg each of HO-1-ARE-luc reporter gene construct and heterologous Nrf2 (pCI-Nrf2) as inducer, pRK5-HA-ubiquitin or its 5 mutants described in A, and FLAG-Arkadia. Representative blots are shown, B. C, HepG2 cells were prepared as in B were blotted for the endogenous target gene hemeoxygenase-1. Representative blots are shown. Values plotted are means ± S.E.M. for duplicate assays from 10 different experiments analyzed by one-way ANOVA. * Statistically different (p<0.05), # statistically different (p<0.0001).