IndraLab

Statements

USP3 binds Flag. 10 / 10
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"To determine the functional consequence of the deubiquitinating activity of USP3 on REST protein turnover, we treated cells with the protein synthesis inhibitor cycloheximide (CHX) in the presence of Flag-USP3 or catalytic mutant Flag-USP3CS."
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"To determine differentially expressed proteins that may serve as potential interaction targets of USP3, total protein extracted from the treated BGC-823 cells stably expressing the Flag-USP3 fusion protein and the BGC-823 cells carrying an empty vector was analyzed using iTRAQ experiments."
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"Moreover, knockdown of USP3 showed a significant reduction in the half-life of REST protein (Fig.  xref G, lanes 5–8), which was then rescued by reconstitution with Flag-USP3 (Fig.  xref G, lanes 9–12) but not with Flag-USP3CS (Fig.  xref H, lanes 9–12)."
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"Similarly, in HEK293 cells, we observed protein stabilization of ectopically expressed Myc-Oct4 as the concentration of Flag-USP3 increased ( xref E), but expression of the catalytic mutant Flag-USP3CS had no such stabilization effect ( xref F)."
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"We co-transfected Flag-USP3 and Myc-Oct4 into HEK293 cells and performed IP with Flag or Myc antibodies, followed by Western blot with reciprocal antibodies."
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"An anti-Flag antibody co-immunoprecipitated ectopically expressed Myc-REST along with Flag-USP3 and vice-versa in HEK293 cells (Fig.  xref A), indicating that USP3 interacts with REST."
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"Flag-USP3 co-precipitated Myc-Oct4 ( xref A), and similarly Myc-Oct4 co-precipitated Flag-USP3 ( xref B)."
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"To validate whether USP3 could increase the REST protein levels, we transfected the neuroblastoma cells with an increasing concentration of Flag-USP3 and catalytic mutant Flag-USP3CS."
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"The increasing concentration of Flag-USP3 showed an increase in endogenous REST protein levels in a dose-dependent manner (Fig.  xref D)."
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"Moreover, the reduced endogenous expression of the REST protein level in USP3-depleted cells was rescued by reconstitution with Flag-USP3 (Fig.  xref J, lane 4 vs. 3)."
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