Figure 4

J19-1 directly targets Keap1 at C171.

(a) Domain structures of human Keap1. Locations of all cysteines and important binding partners are shown and four key cysteines (C151, C171,C273 and C288) are highlighted in red (human Keap1). (b) Total lysates of Keap1-Flag transfected 293 T cells were incubated with Probe or NC at 4 °C overnight. The precipitates resolved by SDS-PAGE were stained by silver staining. (c) The recombinant Keap1 protein was incubated with Probe or NC at 37 °C for 1.5 h and the mixtures were blotted for biotin or Keap1. (d) The recombinant Keap1 protein were incubated with Probe in the absence or presence of a 2–3 fold excess of unlabeled J19-1 for 1.5 h at 37 °C and the mixtures were blotted for biotin or Flag. (e) MS/MS analysis of the recombinant Keap1 incubated with or without J19-1 for 1.5 h. The red arrow indicates the cysteine bound to J19-1. (f) Recombinant wild-type (WT) Keap1 and its mutants were incubated with Probe or NC at 37 °C for 1.5 h, followed by blotting for biotin and Keap1.