Figure 4
Honokiol prevented the TNF-α-induced IκBα proteosomal degradation.
(A) Western blot analysis of IκBα, phospho-IκBαand tubulin (loading control) protein expression in ECs pre-treated with DMSO or honokiol (3 μM) for 30 min, then stimulated with TNF-α (10 ng/ml) at different times (5, 10, 15 and 30 min). (B) IκBα and phospho-IκBαsignals were quantified, expressed as a relative ratio (normalized with tubulin) and plotted against time. (C) Western blot analysis of phosphor-IκBα, IκBα and tubulin protein expression in ECs treated with MG132 (5 μM) for 1 h, then incubated with DMSO or honokiol (3 μM) for 30 min and stimulated with TNF-α (10 ng/ml). (D) Phospho-IκBα and IκBα signals were quantified, expressed as a relative ratio (normalized with tubulin) and plotted against time. (E) EC cells were exposure to DMSO or the GABAA receptor antagonist, SR95531 (20 μM), for 30 min and sequentially treated with DMSO, honokiol (3 μM), or GABAA agonist, muscimol (100 μM) for 30 min. The ECs were stimulated with TNF-α (10 ng/ml) for 15 min and analyzed using western blot. The IκBα signals were quantified expressed as a relative ratio (normalized with tubulin). Representative images from 3 experiments are shown. Data are mean ± SEM from 3 independent experiments. *P < 0.05; **P < 0.01 versus untreated control cells. All the Western blotting experiments were performed under the following condition. After transferring the blots onto nitrocellulose membranes, we immediately cropped the targeted blots according to referenced indicating markers and then targeted proteins were immunoblotted with its specific monoclonal antibody.
