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. 2023 Feb 28;90:104480. doi: 10.1016/j.ebiom.2023.104480

Fig. 5.

Fig. 5

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. β-actin was used as the loading control. (b) Immunoblots for Nrf2 and β-TrCP in whole cell lysates from PMs treated with IR-61 for 24 h after transfection with siCtrl or β-TrCP siRNA (siβ-TrCP). β-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.