Blockade of GSK-3β decreases NF-κB transcriptional activity. BV-2 cells were preincubated with vehicle or TWS119 (2.5 μM) for 30 min before stimulation with 100 ng/ml LPS for the various times indicated. Whole cell lysates and nuclear extracts were prepared. (A) IκB-α degradation is not regulated by GSK-3β. Western analysis was used to determine total and phosphorylated IKKα/β and IκB-α proteins in whole cell extracts. (B) Localization of p65 to the nucleus was determined by immunoblotting. TFIIB immunoblotting was performed to monitor loading. (C) ELISA-based measurement of p65 DNA binding was analyzed as described in Methods. (D) Cells were transfected with a 3XNF-κB-luciferase reporter construct. 24 h post-transfection, cells were preincubated with vehicle or TWS119 (2.5 μM) 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 < 0.05; **p < 0.01 compared with LPS alone.