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发布于:2020-11-27 09:02:11  访问:0 次 回复:0 篇
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Monoubiquitination of eHAX and HAX in the chromatin fraction under normal conditions was not disturbed by the depletion of TIP. Wildtype eHAX was also puried from TIP knockdown cells. Proteins were analyzed by immunoblotting using antiHAX antibodies.The polyubiquitination of eHAX peaked sharply to min after IR and nearly paralleled its acetylation. The similarities in the kinetics of acetylation and of the mono and polyubiquitination of HAX strongly suggest a preferential linkage of acetylation with de novo polyubiquitination of eHAX in the chromatin fraction immediately after IR.Notably, the ratio of polyubiquitination to monoubiquitination of HAX in the nuclear soluble fraction is much higher than that in the chromatin fraction.This suggests that the polyubiquitination but not the monoubiquitination of HAX is involved in the release of HAX from chromatin upon DNA damage.Collectively, these ndings suggest that TIP facilitates HAX release upon DNA damage via acetylationdependent polyubiquitination of HAX at the very early stage of DNA repair.Cells were treated with sodium butyrate for the detection of acetylation.To investigate this further, we examined the modication status of a phosphorylation mutant of HAX after IR.Immunoblotting analysis using antiHAX and antiubiquitin afnity puried from both the nuclear soluble and chromatin fractions were mono and polyubiquitinated following IR. These ndings suggest that phosphorylation is not required for either acetylation or ubiquitination after IR.Consistently, in contrast to what was seen for the acetylation and polyubiquitination of eHAX, which peaked to min after IR, the level of phosphorylation was constant during the experiment. Taken together, these results indicated that acetylationdependent ubiquitination is regulated independently of the phosphorylation of HAX upon DNA damage.The expression of the Targetmol‘s Dicumarol GFPHAX wild type and GFPHAX mutants was not affected by the coexpression of HAX siRNA. These data suggest that along with HAX phosphorylation, HAX acetylation and ubiquitination play a signicant role in cellular survival following DNA damage.In our previous study, we suggested that UBC can regulate the ubiquitination of HAX after IR. Indeed, we found that UBC is included in eHAX complexes that were afnity puried from either nuclear soluble or chromatin fractions and that the amount of UBC in the HAX complexes was substantially increased following IR. We found that both GFPTIP and GFPUBC accumulated at damaged sites min after microirradiation. We conrmed that the integration of UBC into TIP puried from the nuclear soluble fraction is stimulated following IR. Collectively, these ndings indicate that TIP interacts with UBC to facilitate the ubiquitination of HAX in damaged chromatin.This indicated that a fraction of HAX may undergo dynamic release from damaged chromatin.In iFRAP experiments, immediately following microirradiation, all of the uorescence was bleached, and the remaining GFPHAX uorescence was chased using LSM confocal microscopy.As a result, the remaining GFPHAX uorescence within the irradiated area became signicantly weaker than that in the unirradiated area, suggesting that GFPHAX can diffuse from chromatin only in the irradiated area.Thus, DNA damage provokes the release of GFPHAX from chromatin.The dynamics of HAX following DNA damage were examined by determining the uorescence recovery of GFPHAX within two independent strips of a single nucleus, one in the irradiated area and the other in an unirradiated region, immediately following microirradiation, whereas the uorescence intensity in the unirradiated region did not change much during the observed time.
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