Fig. 6. LGK974 inhibits the NF-κB pathway and reduces coimmunoprecipitation of phospho-IκB and β-TrCP in the livers of endotoxemic mice.

Protein and RNA were extracted from the liver tissues of mice injected with 0 or 60 mg/kg LGK974 and 0 or 25 mg/kg LPS. a The levels of the NF-κB pathway components were measured by western blotting. β-Actin and TBP were used as loading controls for total tissue lysates and nuclear lysates, respectively. Complete western blot images are provided in Supplementary Data 5. b–d Band intensities were quantified using ImageJ. e The target DNA binding activity of NF-κB was measured via ELISA. f–i Messenger RNA levels of proinflammatory cytokines were measured by RT-qPCR. Differences among experimental groups were analyzed using one-way ANOVA with Duncan’s multiple range test. The different letters indicate significant differences (^*P < 0.05). j The protein-protein interaction between phospho-IκB and β-TrCP was analyzed by coimmunoprecipitation experiments. The lane marked “Blank” shows coimmunoprecipitation using lysis buffer instead of tissue extract, and the lane marked “Anti-IgG” shows coimmunoprecipitation using an anti-mouse IgG secondary antibody instead of an anti-β-TrCP antibody. k Input samples from the coimmunoprecipitation assay were analyzed by western blotting. IP immunoprecipitation, WB western blot, β-TrCP β-transducin repeat-containing protein.