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Fig. 3. Constitutively active MEK1 enhances the transactivating activity of FOXM1c. (A) Chromatin immunoprecipitation assays of asynchronized BJ1 cells were carried out as described in Methods section to detect FOXM1 binding to the cyclin B1 promoter. Cyclin B1 DNA was enriched more than 50-fold by the anti-FOXM1 antiserum. (B) Schematic diagrams of FOXM1c, FOXM1b and FOXM1c ${Delta}$ Cter. Positions of the DNA binding domain (DBD), exon Va and transactivation domain (TAD) are shown. FOXM1b lacks exon Va. FOXM1c ${Delta}$ Cter was generated by deletion of the last 71 amino acids of FOXM1. (C-E) Transient reporter assays. NIH3T3 cells were co-transfected with the various expression plasmids and cyclin B1 luciferase reporter. 48 hours after transfection, cells were harvested for luciferase assay. (C) 60 ng of FOXM1c, FOXM1b or the control vector pcDNA3 was co-transfected with the cyclin B1 reporter. Both FOXM1c and FOXM1b showed $~$ 2.5-fold stimulation of the cyclin B1 promoter when compared with the vector control. (D) caMEK1 enhances the transactivating activity of FOXM1c. Co-transfection of caMEK1 (30 ng) with an increasing amount of FOXM1 strongly enhanced the transactivating activity of FOXM1c, but not FOXM1b. (E) The caMEK1 enhancing effect requires the presence of functional FOXM1 protein. Various amounts of FOXM1 and MEK1 expression plasmids, and empty vectors (pcDNA3 and pSR ${alpha}$ ), were co-transfected as indicated. Both caMEK1 and functional FOXM1c are required for the synergistic activation of cyclin B1 promoter. (F) Western blot to demonstrate the activating and inhibitory effect of caMEK1 and dnMEK1, respectively, on Raf/MEK/MAPK signaling.

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