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发布于:2020-11-27 09:54:09  访问:0 次 回复:0 篇
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Theyaxis indicates the percentage of each category at each time point.Thus, in human cells, BP focus assembly was induced at quite an early stage in buy RITA response to DNA damage.Hyperphosphorylation of XLBP in cells irradiated with X rays.This retardation was due to phosphorylation, since this slower migration was eliminated by treatment of the immunoprecipitated XLBP with protein phosphatase. The shift was already visible at min postirradiation and became more pronounced at h. XTC cells were preincubated in the presence of either mM caffeine or mM wortmannin for hand then irradiated with X rays.Wortmannin irreversibly inhibits certain members of the PIK family, and mM wortmannin blocks the activities of ATM and DNAPK but not that of ATR in intact cells. We found that the slower migration of XLBP was almost completely eliminated in the extracts prepared from cells irradiated in the presence of mM caffeine. Wortmannin showed a similar effect but was less effective. BP from the untreated HT cells ran slightly more slowly than that from AT cells for unknown reasons.Samples were loaded in an order to see the shifted and the nonshifted bands next to each other.Half of the samples were treated with protein phosphatase. The proteins were run on a gel and immunoblotted with antiXLBP antibodies.XLBP of irradiated cells migrated more slowly than that of nonirradiated cells. Lanes and indicate that the band retardation was already seen at min postirradiation.XTC cells were either untreated or in the presence of mM caffeine. Extracts were prepared from untreated cells, at h postirradiation.To investigate further the possible implication of ATM kinase, we examined the band retardation of BP before and after the irradiation of AT cells that were decient for ATM kinase.The extent of the BP band retardation was signicantly reduced, although the band shift was not completely abolished. Due to technical reasons, the amount of total protein we could load per slot for AT cells was approximately half that of HT cells; therefore, the BP bands were slightly fainter in AT cell samples.Caffeine inhibits the focal redistribution of XLBP in cells irradiated with X rays.Given that the postirradiation phosphorylation of XLBP was reduced in the presence of either caffeine or wortmannin, we were interested in examining whether the focal redistribution of XLBP would also be affected by the presence of these drugs during damage induction.XTC cells were treated as described above, xed, and stained for XLBP. In control cells without inhibitor treatment, the focal redistribution of XLBP was already visible at min postirradiation, and at h, it was prominent. However, in the presence of mM caffeine, the protein stayed distributed mostly homogeneously over the chromatin even after hpostirradiation and the numbers of BP foci assembled were signicantly lower. Wortmannin showed a moderate effect, and the focal redistribution of XLBP appeared to be slightly delayed in the early stages of postirradiation.Later, at hpostirradiation, the assembly of XLBP foci was nearly as efcient as that of the nodrug control. We scored the number of countable BP foci per nucleus by optical sectioning with a confocal microscope.
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