IndraLab

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USP50 binds FlaG. 15 / 15
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"We used the SMART assay xref to measure the degree of exposed ssDNA directly, growing cells in the presence of the nucleotide analogue BrdU before expression, or not of the shUSP50, and FLAG-USP50 variants."
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"In shUSP50-expressing cells given a short exposure to HU, we observed increased lengths of exposed BrdU, which could be suppressed by FLAG-USP50 expression but only partially suppressed by I141R-FLAG-USP50 mutant expression (Fig.  xref )."
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"Moreover, 53BP1 foci were suppressed in shUSP50-expressing cells by additional expression of FLAG-USP50 but not by the I141R-FLAG-USP50 mutant (Fig.  xref )."
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"To address whether USP50 is relevant to the survival of cells experiencing replicative stress, we examined the ability of shUSP50-expressing cells, complemented with FLAG-USP50, to form colonies after exposure to HU."
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"Restart could be improved by FLAG-USP50 expression but not by I141R-FLAG-USP50 mutant expression (Fig.  xref ), indicating a requirement for USP50, and the Ile-141 face in the resumption of replication."
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"To test whether USP50 can bind Ub we expressed FLAG-USP50 and an I141R-FLAG-USP50 mutant along with Myc-tagged Ub (Myc-Ub) and performed FLAG immunoprecipitation."
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"We next generated a FLAG-tagged USP50 construct resistant to that shRNA, which was integrated into an inducible site, expressed upon doxycycline (Dox) treatment, named FLAG-USP50 hereafter."
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"Further, FLAG-USP50 expression improved the number of telomeres, whereas complementation with I141R-USP50 did not (Supplementary Fig.  xref )."
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"In contrast, shUSP50-expressing cells complemented with FLAG-USP50 had replication features comparable to untreated cells, whereas expression of I141R-FLAG-USP50 did not improve replication defects in shUSP50-expressing cells (Fig.  xref ), suggesting a requirement for the Ile-141 face for ongoing replication."
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"We fractioned cells expressing exogenous FLAG-USP50 and noted a proportion of USP50 co-purified with chromatin, which was increased following HU treatment (Fig.  xref )."
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"This method indicated proximity of FLAG-USP50 with both long-label EdU, and short-label EdU/HU treatments (Fig.  xref )."
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"Consistent with Ub-regulated proximity of USP50 to nascent DNA, the signal between FLAG-USP50 and EdU was increased following Ub over-expression and reduced when MG132 was added prior to EdU (Fig.  xref )."
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"The proximity signal between FEN1 and WRN was improved by complementation with FLAG-USP50 but not by I141R-USP50 (Fig.  xref )."
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"We wished to test whether USP50 protein is relevant to replication, despite its low-level expression, and so generated HeLa cells bearing an isopropyl-β- d -1-thiogalactopyranoside (IPTG) inducible shRNA to USP50 (shUSP50-expressing cells), which we demonstrated depleted wild-type, exogenous FLAG-USP50 (Supplementary Fig.  xref )."
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"To examine if USP50 has a role in telomere stability, we grew HeLa cells for several days in telomerase inhibitor, with or without shUSP50 expression, complemented or not with FLAG-USP50 constructs."
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