Fig. 5

Phosphorylation of stathmin1 at Ser16 facilitates phosphorylation of TACC3 at Ser558. A Prediction of potential kinases targeting stathmin1 at Ser16 and TACC3 at Ser558 using the NetPhos 3.1 database. PKA was found to target both of these proteins at the indicated sites. B HCT116 cells were transfected with stathmin1-specific or control siRNAs (final concentrations, 20 nM). Cells were treated with 100 μM bucladesine 4 h before harvesting and harvested 48 h after transfection. The levels of indicated factors were determined by immunoblotting. C Relative levels of expression of the proteins indicated in B. D DLD1 cells were transfected with pCMV-stathmin-FLAG, pCMV-stathmin1-M-S16-Ala-FLAG, or pCMV-FLAG vector (final vector concentrations, 2 μg/ml) and harvested 48 h later transfection. The levels of the indicated factors were determined by immunoblotting. E Relative expression levels of the proteins indicated in D. F DLD1 cells were transfected with vectors as shown in D. Cell viability was measured using MTS assays 0, 24, 48, and 72 h after transfection. G DLD1 cells were transfected with vectors as shown in D. Cell cycle progression was analyzed by flow cytometry 48 h after transfection. H Percentage of cells in G in mitotic phase. I Representative images of tumors formed by DLD1 cells with indicated stable transfections. J The growth curves of the tumors formed by the indicated DLD1 cells (N = 6 mice per group). K Weight differences in tumors formed by the indicated DLD1 cells (N = 6 mice per group)