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. 2006 Aug 3;25(15):3605–3617. doi: 10.1038/sj.emboj.7601243

Figure 2.

Figure 2

(A) COS1 cells were transfected with expression vectors for Keap1-CBD and HA-Nrf2 and the Keap1:Nrf2 complex isolated on chitin beads. The beads were incubated with the indicated peptides, washed, and proteins that remained bound to the beads were analyzed by immunoblot with anti-HA and anti-CBD antibodies. The amounts of peptides added to each sample were 10 ng (lanes 3, 7, 11), 100 ng (lanes 4, 8, 12), 1 μg (lanes 5, 9, 13), and 10 μg (lanes 6, 10, 14). (B) (Upper panel) Thermographs from a representative isothermal calorimetry experiment are shown, in which a 5 μM solution of the Kelch domain was titrated with 50 μM peptide. (Lower panel) The fitted binding isotherms from four experiments are shown. Three experiments (circles, squares, and triangles) were performed by titrating a 50 μM peptide solution into a 5 μM solution of the Kelch domain. A fourth experiment (diamonds) was performed by titrating an 88 μM solution of the peptide into a 6.25 μM solution of the Kelch domain.