Inhibition of GSK-3β activity blocks LPS-induced JNK signaling. BV-2 cells were pretreated with vehicle or TWS119 (2.5 μM) for 30 min and stimulated with LPS (100 ng/ml) for the indicated times. Whole cell extracts or nuclear proteins were isolated. (A) Western analysis was used to determine LPS-induced p38, ERK and JNK activation in the absence or presence of TWS119. (B) Phosphorylated and total c-Jun was detected by western blotting in whole cell extracts. (C) ELISA-based measurement of c-Jun DNA binding was analyzed as described in Methods. Data are presented as mean ± SEM for three independent experiments. *p < 0.05; **p < 0.01 compared with respective LPS-only-treated cells. (D) Cells were transfected with a 3XAP-1-luciferase reporter construct. 24 h post-transfection, cells were preincubated with vehicle or various concentrations of TWS119 for 30 min before stimulation with 100 ng/ml LPS for 6 h. Luciferase activity is presented as a fold of control. Data are presented as mean ± SEM for three independent experiments. **p < 0.01 compared with LPS alone.