2.19. Coimmunoprecipitation (Co-IP) Assay

The Flag or Myc-tagged proteins were immunoprecipitated as we previously described [20]. In brief, the overexpression plasmid of Nrf2, OPTN, and according empty vectors was purchased from Shanghai GenePharma Co., Ltd (Shanghai, China). According to the manufacturer's protocol, HEK-293T cells were transiently cotransfected with the Myc-Nrf2 and the Flag-OPTN vector or empty vector for 48 h. The transfected cells were collected and lysed in immunoprecipitation lysis buffer supplemented with protease inhibitor cocktail. Afterward, the cell lysates were incubated with anti-flag antibody-conjugated agarose beads or antibodies (1-2 μg) for 4 h at 4°C, followed by another 1 h incubation with protein A-Sepharose beads (GE Healthcare, Waukesha, USA) if free antibody was used. Immunoprecipitates were washed, resolved by SDS-PAGE, and then immunoblotted with the appropriate antibodies.