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Not4 proteostasis regulation requires the RRM-C domain. (A) Log phase extracts were prepared from the indicated strains under denaturing conditions, extracts were resolved by 7.5% SDS-PAGE, and then immunoblotted with α-ubiquitin. Blots were stripped and re-probed with α-FLAG and α-G6PDH to control for loading, and the total ubiquitin signal was quantified and normalized to G6PDH levels. Data are representative of four independent experiments. Note that because not4Δ control vector cells consistently express higher amounts of G6PDH, the quantified results underestimate the true increase in global polyubiquitylation in this sample. (B) Cell extracts from the indicated strains were prepared, incubated with the LLVY-AMC fluorescent substrate, and fluorescence quantified using a fluorescent plate reader. Triplicate independent extracts were analyzed per sample and the average and SD are plotted. Statistical significance was determined by pairwise (relative to Not4WT) two-sided Student’s t-test. **p < 0.01; ***p < 0.005; ****p < 0.001. (C) Immunoblot of extracts from (B) using α-Rpt2 (a 19S proteasome subunit) or α-20S. Note that the α-20S antibody recognizes multiple 20S subunits that have nearly identical mass so their signals overlap. (D) Immunoblot analysis of H3K4me3 and total H3 from the indicated strains. The H3K4me3 and H3 signals were quantified and expressed as a ratio. Data are representative of a minimum of four independent experiments. (E) Equal numbers of cells were 5-fold serially diluted and spotted to control media or media containing 0.5 mg/mL azetidine-2-carboxylic acid (AZC) and incubated at 30 °C for six days. (F) As in (E), except cells were incubated on control media or media containing 0.05 µg/mL cycloheximide for two days.
Image collected and cropped by CiteAb under a CC-BY license from the following publication: The conserved RNA recognition motif and C3H1 domain of the Not4 ubiquitin ligase regulate in vivo ligase function. Sci Rep (2018)
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RUO – Research Use Only |
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