FIG. 7.
IL-1β-stimulated redox-active endosomes activate the IKK complex. (A) Strategy for evaluating IKK activation by redox-active endosomes in an in vitro reconstitution assay. (B) IKK assays for kinase activity were performed using in vitro reconstitution of three components: (i) immunoprecipitated IKKα, (ii) isolated vesicles, and/or (iii) GST-IκBα as a substrate. Vesicles and immunoprecipitated (IP) IKK were isolated from untreated or IL-1β-treated MCF-7 cells. Three additional treatments were performed prior to IL-1β stimulation and isolation of vesicles. These included infection with Ad.Dyn(DN) or treatment with SOD and/or catalase proteins. Components I to III were combined, as indicated, in the presence of [γ-32P]ATP. GST-IκBα phosphorylation was evaluated by SDS-PAGE autoradiography (top panel). Western blotting using an anti-GST antibody was then performed to confirm equal loading of GST-IκBα (bottom panel).