It FTY720 Gilenia not to a St Tion of the function MCAK observable at a time 2 expression. Since the FLAG antibody was Body us a lot of st Rkeres signal than the antique Body of MCAK, followed most of the data in this study pr Sentierten FLAG tagged MCAK. However, all results were in non-transfected cells using the antique Rpers MCAK to the best amount to s Problem R that the endogenous proteins To behave Similar way. Ganguly et al. Page 2 cell cycle. Author manuscript, increases available in PMC 2009 1 October. To determine the stability of t the FLAG MCAK, clone 2 was grown without tetracycline for one day to the protein au OUTSIDE the building Rmutter accumulate, then tetracycline was added to prevent the expression further.
The cells were harvested at various time points after the addition of tetracycline, and cell lysates were Western blot for GSK1349572 1051375-16-6 FLAG MCAK and actin content analyzed. Because actin is a stable protein that does not cozy Regulation of the tetracycline was its abundance remained relatively constant and served as control over the time of our experience. In contrast, decreased FLAG MCAK fa Next, and was largely gone by 12 h. The limited stability t of FLAG MCAK led us to ask whether endogenous MCAK one Has similar half-life. To answer this question, we have an antique MCAK body to carry out the same experiment with non-transfected CHO, HeLa and MCF-7 cells, au He that in this case, the cells with puromycin to be treated to stop protein synthesis. The results show that endogenous MCAK disappear in CHO cells T rapidly as MCAK transfected flag, but the protein in HeLa cells and MCF-7 cells seems to be more stable.
Based on this observation, we thought that the stability t of MCAK on the growth rate of the different cell lines dependent Depends. to support this idea, we found that the lifetime of the protein correlated well with the 14 h, 21 h, 38 h and the doubling time, ma s when we tested the growth rates of the three cell lines. The correlation between the rate of cell growth and stability t MCAK in various cell lines have suggested that the protein was degraded in a particular cell cycle. To this M Opportunity to test directly, Clone 2 expressing FLAG MCAK induced and synchronized by the S-phase and sequential BL skirts M phase after release from mitotic block, cells were DNA-F Observed staining microscope the determination distribution of cells in various stages of mitosis.
Flow cytometry data demonstrating the effectiveness of the synchronization is shown in Fig. S1. Western blots were used to create the level FLAG MCAK relative to assess actin in different phases of the cell cycle. The use of non-synchronized cells as a reference, we found that FLAG MCAK levels several times increased Ht the cells in S phase, G2 and mitosis, w During mitosis has accumulated decreased and reached the lowest level of the early G1 or telophase . In accordance with a recently published published shall report, 8 we observed a slow migrating band FLAG MCAK may need during the mitosis. The upper band probably a phosphorylated form of MCAK FLAG, as it can be eliminated by phosphatase treatment mitotic extracts. FLAG MCAK / actin-money ratios were used for each phase of the cell cycle and the calculated data are shown in Fig. 3B. The figure compares the total amount MCAK FLAG compared to all phases of the cell cycle, but it should be observed in the vest