, GABA inhibition the upper band disappeared faster than the lower band, which is preferably reduced to the M Possibility that a phosphorylated form of FLAG MCAK k Nnte. Are similar results as those obtained in non-transfected cells was compared using an antique Rpers MCAK. Measurements of biochemical degradation MCAK in different phases of the cell cycle by the fact that the cells not by mitosis perfect match move complicated. For example, the cell population as M / T marked in. Prometaphase 40%, 35% in the north he metaphase, anaphase 5%, 15% and 5% telophase in the early G1-3B cells, we have the F staining the chromosomes to the following stages of mitosis business included protected.
In an attempt to provide a more accurate indication of when MCAK is w To get degraded during mitosis, we turned to a microscopic analysis, in mitotic cells with an antique Body against FLAG MCAK Ganguly et al found Were rbt. Page 3 of the cell cycle. Author manuscript, increases available in PMC 2009 1 October. and photographed, so that cells GSK1838705A 1116235-97-2 can be directly compared in various stages of mitosis with a constant exposure. As shown in Fig. 4, prophase cells usually had the bright F FLAG staining and showed MCAK localized to p The spindle, the kinetochores, and some of the microtubules starting from p Of time. F Staining remained strong in prometaphase but was significantly lower in metaphase, anaphase, telophase and. Although the F Staining was significantly reduced in the late stages of mitosis, it was is not completely absent, as indicated by the improved image of the inset in Fig.
4C. Note the improved picture, however, that the kinetochores are barely visible, and even Polf Staining is only slightly above the low color normally associated with microtubules. As a contr For the experiments, the cells were also treated with an antique Body, the kinetochore CREST-F Staining at the cell No. cycle.17 decrease CREST-F Staining in anaphase or telophase noted compared with prometaphase f Rbte. Although immunofluorescence is not between the degradation of MCAK and MCAK dissociation of binding sites, the fact that the loss of F coloring Difference occurred approximately at the same time, we measured a decrease in levels of MCAK biochemically, claimed that there Degradation occurs Haupts chlich in metaphase to anaphase transition.
Immunofluorescence of untransfected cells with an antique Body against k Rpereigene MCAK also exhibited a significant reduction associated MCAK kinetochore in anaphase and telophase. The preferential loss of the slow migration band w Proposed during mitosis, the M Possibility that phosphorylation of MCAK its degradation products to foreign St. A number of supply Publications have shown that the kinase Aurora B phosphorylates MCAK may need during the mitosis and regulates its function. To test whether phosphorylation by the kinase Aurora B also the signal degradation MCAK we first is an examination of the effectiveness of cloning to the minimum lethal dose of the inhibitor of the kinase Aurora to determine B ZM447439 in CHO cells 0.3 g / ml . Then clone 2 cells synchronized with thymidine and release of cells in the media with nocodazole in the presence or absence of ZM447439 at concentrations of 1.7, 3.3 and 6 times the minimum lethal dose. At all concentrations, the addition of ZM447439 no effe