Fig. 3

(A) Phylogenetic tree for the BTB domain of Keap1 proteins. The numbers associated with the branches indicate bootstrap values. (B to E) Ratio of Keap1A and Keap1B mRNAs at the indicated developmental stages in O. latipes (B), D. rerio (C), X. laevis (D), and X. tropicalis (E). Data are derived from RNA-seq analysis in previous studies (50–53). (F) Alignment of amino acid sequences for the BTB domain of Keap1 proteins. Keap1A- or Keap1B-specific amino acids are highlighted in red or blue, respectively, with common amino acids shown in brown. (G) Domain organization of zKeap1A, zKeap1B, and the C3IR mutants zKeap1A(A→B) and zKeap1B(B→A) as well as a summary of their interaction with zCul3 as determined in (H) and (I). (H and I) Lysates of HEK293T cells expressing FLAG-tagged zKeap1A, zKeap1B, zKeap1A(A→B), or zKeap1B(B→A) together with HA-tagged zCul3A (H) or zCul3B (I) were subjected to immunoprecipitation with antibodies to FLAG. (J) Domain organization of human Keap1 and the C3IR mutants hKeap1(W→3), hKeap1(W→A), and hKeap1(W→B) as well as a summary of their interaction with human Cul3 as determined in (K). (K) Lysates of HEK293T cells expressing FLAG-tagged hKeap1, hKeap1(W→3), hKeap1(W→A), or hKeap1(W→B) together with HA-tagged hCul3 were subjected to immunoprecipitation with antibodies to FLAG. (L and M) Lysates of HEK293T cells expressing HA-tagged hCul3 together with FLAG-tagged hKeap1 or its exchange mutants containing C3IR of Keap1 proteins from the indicated species were subjected to immunoprecipitation with antibodies to FLAG (L). The relative extent of HA-hCul3 binding to the hKeap1 mutants was also quantitated by densitometry (M). Quantitative data are means from three independent experiments.