2.9  Ubiquitination assay
To detect ubiquitin-conjugated endogenous Keap1, cells were treated with or without 80 nM of digoxin for 6 h. The cell lysate was incubated with Keap1 antibody overnight at 4 °C, and then incubated with protein A/G-agarose beads (Santa Cruz, Texas, USA) for another 4 h at 4 °C. Immunoprecipitated proteins were analyzed by western blot with ubiquitin antibody. The band intensities of ubiquitinated Keap1 was quantified using Image J software.