Figure 3. PRIMA-1^Met inhibited kinase activity of MEK in vitro and in cells.

A. Representative results of in vitro kinase assay. Inactive ERK1 protein was used as a substrate and mixed with active MEK kinase and different doses of PRIMA-1^Met. The phosphorylation level of ERK1 (Thr202/Tyr204) was detected by Western blot analysis. Total MEK was used as loading control and MEK inhibitor AZD6244 was used as a positive control. B. Western blot analysis of MEK protein expression level in six colorectal cancer cell lines and normal colon epithelial cell line FHC. The activation of MEK signaling was detected in HCT116^neg C., HCT116^wt D. and SW480 E. cells. After starvation in serum-free medium for 24 h, cells were treated with different doses of PRIMA-1^Met for 12 h and then treated with 5 ng/ml EGF for 30 min. AZD6244 was used as a positive control. Data were representative results from three independent experiments.