To further validate the dependence of this reporter on the 26S proteasome, siRNAs were used to deplete selected components of the 20S core particle and 19S regulatory complex in MDA-MB-231-4xUb-Luc MDA-MB-231-4xUb-Luc MDA-MB-231-4xUb-Luc MDA-MB-231-4xUb-Luc MDA-MB-231-4xUb-Luc
Moreover, this illustrates that even highly unstable mRNAs are further destabilized by SOX, and furthermore, that the failure of SOX to deplete select transcripts is not a non-specific effect of enhanced competition for or saturation of the decay machinery [11].
Experiments with PBMC depleted of selected cell subsets or with purified single cell subsets revealed that CD64+ monocytes were necessary and sufficient to confer massive CD4 down-modulation of BT-061 decorated T cells.
To identify the immune cell subset that conferred CD4-down modulation of BT-061 decorated T cells, co-incubation experiments were performed either with PBMC depleted of selected immune cell subsets, or with the respective MACS-enriched immune cell subsets.
In each iteration, the input promoter set is depleted by the selected promoter, plus those promoter sequences that scored below a predefined threshold (see Materials and Methods).
Libraries were then prepared from the now rRNA depleted, fragmented, size selected, and kinased RNAs using the NEBNext Small RNA Library Prep Set according to the manufacturers protocol (New England BioLabs).
Finally, the growth rate of 16 strains, carrying the same marker and supplement combination that replicates the 16 metabotypes (Mülleder et al., 2012) did not distinguish the depleted from the selected metabotypes either.
To select shRNAs that were depleted after selection we calculated the pre-/post-ratio and repeated the above procedure.
To select cells depleted of TAPBPR puromycin selection was used post transduction with V2LHS_135531.
About 50 chromosomes assembled in control extracts or depleted extracts were randomly selected from three independent experiments.
