Fig 2: IR-61 binds with Keap1 Protein. Recombinant human Keap1 protein was incubated with IR-61 in 1 M HEPES buffer (pH 7.4) at 37 °C for 30 min, and then the mixture was subjected to western blot. NIR fluorescence band was detected (left panel) and then incubated with Keap1 antibody to scan the protein band (right panel).

Fig 3: IR-61 promotes Nrf2 stabilization through competitive binding with keap1. (a) Immunoblots for Nrf2 in whole cell lysates from the PMs incubated in the presence or absence of 5 mM NAC for 4 h and then treated with 10 µM IR-61 for the indicated time. ß-actin was used as the loading control. (b) Immunoblots for Nrf2 and ß-TrCP in whole cell lysates from PMs treated with IR-61 for 24 h after transfection with siCtrl or ß-TrCP siRNA (siß-TrCP). ß-actin was used as the loading control. (c) SPR assay for interaction of IR-61 with Keap1 protein. (d) Immunoblots for Keap1 in whole cell lysates from PMs incubated in the presence (+) or absence (-) of 10 µM IR-61 at different temperatures. (e) Keap1 band intensity was quantified by ImageJ. Blot intensity was normalized to intensity obtained for the 37 °C sample. (f) SPR assay for Nrf2–Keap1 interaction treated with 0, 2.5, 5, and 10 µM IR-61. (g, h) PMs were treated with IR-61 or vehicle for 24 h. Protein levels of Keap1 and Nrf2 as well as the interaction between Keap1 and Nrf2 were assayed using western blot and co-IP. (i) Ligand docking analysis for IR-61 with human Keap1 protein.

Fig 4: IR-61 has no effect on Keap1 expression or Keap1-p62 interaction. PMs were treated with IR-61 or vehicle for 24 h. The interaction between Keap1/p62 was determined by co-IP assay.

Fig 5: Activation of Nrf2 in macrophages at infection sites imparts protection against sepsis. IR-61 selectively accumulates in macrophages at infection sites upon i.v. administration. Then IR-61 activates Nrf2 mainly by directly inhibiting the Keap1–Nrf2 interaction, which further enhances antibacterial defence of macrophages at infection sites and treat sepsis precisely.