Shikonin Suppresses Human T Lymphocyte Activation through Inhibition of IKKβ Activity and JNK Phosphorylation
Figure 6
Effect of shikonin on inhibition of IKK-β activity (a) and IKK-α/β phosphorylation (b). Human HEK293 cells transfected with FLAG-IKK-β (wt) plasmid were immunoprecipitated (IP) with anti-Flag antibody, and the IP Flag-IKK-β was incubated with GST-IκBα substrate and ATP in the presence or absence of 100 μM shikonin. IKK-β kinase activity was determined by the level of phosphorylated GST-IκBα using antibody against p-IκBα (a). The human T lymphocytes were pretreated with shikonin at 37°C for 60 min and then stimulated with PMA (20 ng/mL)/ionomycin (1 μM) (P/I) at 37°C for different time points. The whole-cell lysates were prepared, and proteins were analyzed by Western blotting using antibodies against IKKα/β and the phosphorylated form of IKKα/β. Data are representative of three independent experiments.
