2.12. Co-immunoprecipitation

To detect the influence of DSF/Cu on the interaction between NRF2 and Keap1, cells transfected with exogenous Keap1 expression vector were treated with DSF/Cu for 12 h, then lysed with IP lysis buffer supplemented with protease and phosphorylation inhibitor cocktail for 60 min on ice, and centrifuged at 12,000 g for 10 min at 4 °C. The supernatants were incubated with Keap1 or Isotype IgG antibody overnight at 4 °C, following the co-incubation with protein A/G conjugated beads for 2 h at room temperature. Beads were washed with TBST three times, and then boiled in SDS-loading buffer and subjected to Western blot.