Shikonin Suppresses Human T Lymphocyte Activation through Inhibition of IKKβ Activity and JNK Phosphorylation
Figure 5
Effect of shikonin on inhibition of nuclear translocation of NF-κB subunit p65 (a), degradation and phosphorylation of IκB-α in human T lymphocytes stimulated by PMA/ionomycin (b and c). For analysis of the intercellular NF-κB expression, cells were incubated with shikonin for 60 min, and then fixed immediately by cytofix buffer after costimulation by PMA (20 ng/mL)/ionomycin (1 μM) for 120 min, stained with NF-κB antibody for 60 min avoiding light, and then analyzed by flow cytometry. The unstimulated cells were served as negative control (a). For detection of IκB-α, cells were incubated with or without shikonin for 60 min (b); for detection of pIκB-α, the human T lymphocytes were pretreated with or without shikonin and 100 μM ALLN for 60 min (c) and then stimulated with PMA (20 ng/mL)/ionomycin (1 μM) (P/I) at 37°C for 60 min. The whole-cell lysates were prepared, and the proteins were analyzed by Western blotting using antibodies against IκB-α and P-IκB-α. Data are representative of three independent experiments.
