Fig. 4.

Effect of DHGA-D on MLK3 activity and binding. (A) DHGA-D suppresses MLK3 activity. MLK3 kinase assays were performed as described in the Materials and Methods. Data are presented as the means ± SD of three independent experiments. Asterisks [(**) and (***)] indicate significant differences [(P < 0.01) and (P < 0.001), respectively] between groups with or without DHGA-D. (B) DHGA-D directly binds to MLK3. Binding affinity between DHGA-D and MLK3 was confirmed in vitro by immunoprecipitation assay with DHGA-D Sepharose beads, followed by immunoblotting by using an anti-MLK3 antibody as described in the Materials and Methods. First lane: input control, MLK3 protein; second lane: (control), MLK3 protein precipitated with Sepharose 4B beads; third lane: MLK3 protein precipitated with DHGA-D Sepharose 4B beads. Each experiment was performed three times.