Fig. 2. The MLK inhibitor CEP-11004 blocks MLK3-induced phosphorylation of golgin-160. (A) HeLa cells were transiently transfected with Myc-tagged golgin-160 in the absence or presence of MLK3 and then treated in the absence or presence of CEP-11004 (0.5 µM) during the last 5 hours of the experiment. Protein lysates were separated by SDS-PAGE for 20 hours at 20 mA to achieve separation of phosphorylated forms of golgin-160 (top) and MLK3 (bottom). The gel shift of MLK3 is due to autophosphorylation. (B) CEP-11004 blocks MLK3 but not active DLK. Cells were transfected with HA-tagged JNK1 alone or in the presence of DLK or MLK3 followed by treatment with or without 0.5 µM CEP-11004. Lysates were immunoblotted for phosphorylated JNK1 (pJNK), HA-JNK1, DLK (Flag tag) or MLK3 expression. The activation level of JNK shown in the lower graph was determined by densitometry as described in Materials and Methods. (C) CEP-11004 does not inhibit MKK1. Cells transfected with wild-type or inactive (KR) MLK3 or active MKK1 were treated in the absence or presence of CEP-11004. Lysates were immunoblotted for active ERK (ppERK, top) or -tubulin (bottom) as a protein loading control. The lower graph shows densitometry analysis of the relative amounts of ppERK in each condition. The activation level of ERK was determined by densitometry, as described in Materials and Methods. Data represent two separate experiments.