Fig. 4

Identification of novel small molecular compounds targeting IκBα and modulate ROS. a Space filling representation of the ankyrin repeat domain (in magenta) bound to a partially truncated NF-κB heterodimer (p50/p65, in cyan and in green respectively), pdb code 1NFI. b 3D structure of the IκBα and p65 interaction with the identification of the pocket binding domain. c Best pose of ZINC26500814 (Psammaplin-A) in the site of binding located in the task1 of p65. d IC-50 values of PSA in A549 cells lines treated for 48 h and 72 h and analyzed by CTG assay. e Immunofluorescence on A549 cells treated with 5 μM PSA for 1 h and 2 h was performed to detect localization of endogenous p65 (red signal) and IκBα (green signal). Nuclei were stained with DAPI (blue). Yellow arrows indicate changing p65 localization. f IC-50 values of PSA in H460 cells lines treated for 48 h and 72 h and analyzed by CTG assay. g Immunofluorescence on H460 cells treated with 5 μM PSA for 1 h and 2 h was performed to detect localization of endogenous p65 (red signal) and IκBα (green signal). Nuclei were stained with DAPI (blue). Yellow arrows indicate changing p65 localization. h Immunoprecipitation of MYC from HEK293T transfected with a MYC-IκBα and FLAG-p65; FLAG and MYC were detected by Western blotting. The IκBα/P65 inhibitor, PSA, was added to HEK293T for 1 h. Total extract (TE) was performed as a control and was immunoblotted with, FLAG, MYC and Vinculin for loading controls. i and j Immunoprecipitation of endogenous IkBα in A549 and H460 cells, untreated or treated with 5 μM PSA for 1 h; p65 was detected by Western blotting. Total extract was used as a control and was immunoblotted with, p65, IκBα and Vinculin as loading control. k Western blotting of GST pulldowns showing that IκBα loses its interaction with p65. A549 cells were lysed, and GST pulldown assays were carried out by incubating lysates with immobilized purified, bacterially expressed GST- IκBα in the presence or absence of 100 μM PSA for 1 h. Pull-down fractions were subjected to Western blotting using anti-p65 antibodies