(b) Immunoprecipitation assays. Flag-tagged Keap1 was co-expressed with increasing concentrations of green fluorescent protein (GFP)-p62 (lanes 3-6) in HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody. The resulting immunoprecipitates were subjected to SDS-PAGE and analysed by immunoblotting with anti-Flag, anti-p62 and anti-Nrf2 antibodies. The bands corresponding to Flag-Keap1, endogenous p62, Nrf2 and actin are indicated. Data are representative of three independent experiments. 