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Issues to be addressed: 1) The histone turnover experiment would benefit from the inclusion of total H3 control ChIP in WT and mutant backgrounds.
Reviewer #3: 1) The histone turnover experiment would benefit from the inclusion of total H3 control ChIP in WT and mutant backgrounds.
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In single turnover experiments using plasmid substrates, I-SceI generates transient open circle intermediates during the conversion of supercoiled to linear DNA, indicating that the enzyme cleaves the two DNA strands sequentially.
We also demonstrate how multiple turnover experiments can provide evidence for either "burst-phase" kinetics, which can reveal a slow product release step, or linear-phase kinetics, in which the chemical step can be rate-determining.
Turnover experiments were performed using RGC5 cells, as well as the low and high expressers.
Turnover experiments showed that the endogenous optineurin was relatively short-lived, with a half-life of approximately 8 hours.
By staining the recycling vesicle pool by performing FM styryl dye turnover experiments, we found a heterogeneous distribution of the number of neighbours for each bouton (see Figure 1C), resulting in areas of varying synapse densities.
To determine whether the RanBP9-SSH1 complex alters SSH1 protein stability, we performed cycloheximide turnover experiments.
In protein turnover experiments, cells were treated with 50 μg/ml CHX for 0 8 hr prior to lysis.
For turnover experiments with p97/VCP QQ-HA, DNA construct was co-transfected with Myc-tagged rhpdopsin plasmid DNA followed by the inhibition of protein synthesis with cycloheximide.
RNA turnover experiments show that the greater mRNA levels upon cohesin depletion are caused by increased transcription not increased RNA stability (Schaaf et al. 2009).
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