Association of MLK3 and GSK-3β. BV-2 cells were pretreated with vehicle or TWS119 (2.5 μM) for 30 min prior to LPS treatment for the indicated times. Cell lysates were immunoprecipitated (IP) with goat anti-MLK3 antibody, and the resulting precipitates were separated by SDS-PAGE in the presence (A) or absence (B) of the reducing agent DTT. Immunoblotting was performed using rabbit anti-GSK-3β antibody or rabbit anti-MLK3 antibody. The cell extracts were also blotted for MLK3, GSK-3β and β-actin. The band intensity of MLK3 dimers was quantified with a densitometric analysis, and calculated as the optical density × area of band.