Figure 6

SP600125, a JNK inhibitor, mimics GSK-3β inhibitor action in LPS-treated BV-2 cells. Cells were pretreated with vehicle or SP600125 (SP, 20 μM) for 30 min followed by treatment with LPS (100 ng/ml) for the indicated times. Whole cell extracts or nuclear proteins were isolated. Western blotting (A) and ELISA-based measurement of c-Jun DNA binding (B) were analyzed as described in Fig. 5. (C) AP-1 luciferase reporter assays were performed by LPS stimulation in the absence or presence of SP600125 (6 h) of BV-2 cells. (D) Cells were pretreated with various concentrations of SP600125 for 30 min followed by exposure to 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. **p < 0.01 compared with respective LPS-only-treated cells in (B) or compared with LPS alone in (C) and (D). The TNF-α content in untreated cultures was not detectable. The level of TNF-α in cells treated with LPS alone was 4.22 ± 0.25 ng/ml.