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发布于:2020-11-27 17:46:03  访问:0 次 回复:0 篇
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These observations indicated that BAK interacted with MCL in uninfected cells, which was supplanted by an interaction between EB K and BAK upon infection.No coimmunoprecipitation of BAK with BCL, BCLXL, BIM, BID, or tBID was observed in uninfected or infected cells. BAK and MCL form a complex that is disrupted during sell TBTA adenovirus infection.Western blotting was carried out on precipitated material with an antiMCL antibody, antiBAK plus antiBAX antibodies, or an antiEB K antibody.Samples of lysates collected before IP were analyzed to ascertain total protein levels prior to immunoprecipitation.EA expression during adenovirus infection causes the loss of MCL protein.Adenovirus infection stimulates proteasomemediated turnover of MCL.The effect of adenovirus infection on mcl mRNA levels.Fold change represents the difference in fluorescence signal in the and h samples compared with the corresponding h sample.Error bars, the standard deviation of triplicate samples.In infected cells expressing EA, loss of mcl mRNA is progressive over the course of infection, exhibiting up to sixfold repression. Levels of gapdh mRNA also decreased during infection, but significantly less than mcl mRNA. All three sets of transfected cells were GENES DEVELOPMENT translation inhibitor cycloheximide. Inhibition of new protein synthesis under CHX treatment reduced the levels of MCL protein modestly in mockinfected cells, which was more pronounced in adenovirusinfected cells. Addition of EPO for either or hprevented much of the loss of MCL protein in both infected and uninfected cells, indicating that MCL protein turnover is regulated by ubiquitindependent proteasome degradation.In mockinfected cells, MCL was relatively stable with an apparent halflife h.In contrast, adenovirus infection dramatically increases the rate of MCL protein turnover with an apparent halflife of h, which is reversed by addition of a proteasome inhibitor.Levels of actin remained constant under all conditions.These observations indicated that adenovirus infection stimulates the specific turnover of MCL, suggesting a mechanism for initiation of cell death during adenovirus infection that is upstream of EB K inhibition.The percentage of cells undergoing apoptosis in the transfected population was determined visually by counting the number of condensationpositive cells in transfected cells per sample.These observations showed that MCL was functionally capable of inhibiting adenovirusinduced apoptosis, and suggest that under normal conditions, endogenous MCL must be lost in order for adenovirusinduced apoptosis to occur.To test whether MCL downregulation is sufficient for exposure of this BAK epitope indicative of conformational activation of BAK, we utilized RNA interference to downregulate MCL.BAKMCL coimmunoprecipitation was observed with the BAKTM antibody as expected. Thus, MCL downregulation by EA may be required for apoptosis, but it is not sufficient for BAK activation and cell death.Exogenous MCL overexpression rescues apoptosis in adenovirusinfected cells.Rhoadmine stain represents cells expressing the transfected gene, whereas blue stain represents chromatin staining.White arrows indicate the corresponding chromatinstained cells from the matching rhodaminestained micrograph.MCL expression rescues infected cells from adenovirusinduced apoptosis in the absence of EB K.Error bars, the standard deviation of counts from two separate experiments.Downregulation of MCL is not sufficient for BAK activation.ATM responds to doublestrand DNA breaks by undergoing autophosphorylation at serine, and a downstream substrate in the ATM pathway, HAX, becomes phosphorylated on serine.
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