Figure 2. KEAP1 interacts with IKKβ via KEAP1 Kelch domain.
(A) Depletion of endogenous KEAP1 by KEAP1 siRNA led to accumulation of IKKβ but not of other components involved in NF-κB activation.
(B) Exogenous interaction between KEAP1 and IKKβ. Lysates of HEK-293T cells cotransfected with HA-tagged KEAP1 and Flag-tagged IKKβ.
(C) Endogenous interaction between KEAP1 and IKKβ in both unstimulated and TNFα–stimulated MDA-MB-435 and HEK-293 cells.
(D) KEAP1 directly interacted with IKKβ. In vitro transcribed and translated [35S]methionine-labeled KEAP1 proteins were incubated with recombinant His-tagged IKKβ proteins, pulled down by Ni2+ beads, separated by SDS-PAGE, and visualized by autoradiography. IVT, in vitro transcription and translation.
(E) The effect of KEAP1 knockdown on TNFα–mediated IKKβ activation.
(F) Interaction between the KEAP1 Kelch domain and IKKβ as examined by co-IP. Schematic shows five different domains of KEAP1, including an N-terminal region (amino acids 1–60), a BTB domain (amino acids 61–179), a BACK domain (amino acids 180–314), a Kelch domain (amino acids 315–598, six Kelch motifs), and a C-terminal domain (amino acids 599–624).
(G) Mammalian-two hybrid assay revealed the direct interaction between KEAP1 Kelch domain and IKKβ. The indicated regions of KEAP1 fused to the GAL4 DNA-binding domain (DBD) were cotransfected VP16 activation domain (AD) –fused IKKβ (full length), GAL4 luciferase reporter, and TK-rLuc reporter (internal control). Error bars represent SDs (n=3).