IndraLab

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Flag binds OTUB1. 25 / 25
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"This indicates that the expression of Flag-OTUB1 stabilizes HA-NS2."
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"HEK293T cells were cotransfected with plasmids encoding subunits of RdRp (pHW-PB2, pHW-PB1, and pHW-PA), pHH-NA, pHW-NP, pNS2, pFlag-OTUB1, or pcDNA3.1(+) as indicated in xref ."
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"Although transfecting pFlag-OTUB1 did not influence transcription as activated by RdRp ( xref , column 3), transfecting the cells with pNS2 decreased transcription by 25% ( xref , column 4)."
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"Transcription activation declined by 30% if Flag-OTUB1 was also expressed ( xref , column 5)."
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"Interestingly, the amount of cRNA increased by 1.7-fold after the cells were cotransfected with pNS2 and pFlag-OTUB1 ( xref , column 10)."
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"The result showed that OTUB1 was readily detected in the immunoprecipitated complexes of Flag-SLC7A11, similarly, SLC7A11 was co-immunoprecipitated with Flag-OTUB1 ( Fig. 5 D)."
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"After the cells were also transfected with RdRp and NP plasmids ( xref , column 12) or RdRp plasmids and pFlag-OTUB1 ( xref , column 13), the amount of NA vRNA in the cells increased slightly."
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"NA vRNA levels increased by 1.6-fold after cotransfection of RdRp plasmids, pHW-NP, and pNS2 ( xref , column 14), and further increased by 2.2-fold after pFlag-OTUB1 was additionally transfected ( xref , column 15)."
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"A reciprocal co-immunoprecipitation assay was then performed by co-transfection of Nur77 and Flag-OTUB1 into HeLa cells, and the result further validated the interaction of Nur77 and OTUB1 ( Fig. 2 C)[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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"When Flag-tagged WT OTUB1 (Flag-OTUB1) was cotransfected with empty vector, Myc-SET7 or its enzymatically deficient mutant (Myc-SET7-H297A) ( xref ) in OTUB1 −/− H1299 cells, monomethylation of OTUB1 in the cells transfected with WT SET7 was higher than in the cells transfected with empty vector control, while monomethylation of OTUB1 in the cells transfected with Myc-SET7-H297A was similar to the cells transfected with empty vector control, suggesting that OTUB1 might be methylated by endogenous SET7 in these cells or OUTB1 might be methylated at K122 by another unknown methyltransferase ( xref E )."
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"Furthermore, in an immunoblot analysis, we showed that the expression of Flag-OTUB1 increased the abundance of NS2 ( xref )."
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"Additionally, transfecting the cells with pNS2 or both pNS2 and pFlag-OTUB1 followed by IAV infection fully restored vRNP export ( xref , Bar chart, columns 3, 4), suggesting that siRNA knockdown of OTUB1 hampers the nuclear export of vRNP."
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"In addition, vRNP nuclear export was fully recovered after the expression of NS2 and Flag-OTUB1 ( xref , column 4)."
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"Next, we co-transfected Myc-tagged CST1 and Flag-tagged OTUB1, the results showed that in the absence of OTUB1, CST1 could bind to GPX4 (Fig. xref , lane 2); in the absence of CST1, OTUB1 could also bind to GPX4 (Fig. xref , lane 3); and when OTUB1-Flag was transfected at the same time, OTUB1 bound to GPX4 was significantly increased upon CST1-Myc (Fig. xref , lane 4)."
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"Transfection of pFlag-OTUB1 was shown to increase the stability of HA-NS2 ( xref ), thereby confirming that OTUB1 can deubiquitinate and stabilize NS2."
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"Subsequently, HEK293T cells were transfected with CST1-FL/N/C-Myc plasmids and OTUB1-Flag, GPX4-HA plasmid, respectively."
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"Transfection of Flag-OTUB1 into breast cancer cells with OTUB1 knockdown led to the restoration of ERRα expression in MDA-MB-231 cells, as demonstrated in xref L. It is also noteworthy that the overexpression of OTUB1 promoted ERRα translocation, whereas the knockdown of OTUB1 repressed it ( xref E-H)."
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"To verify the interaction between NS2 and OTUB1, we cotransfected HEK293T cells with plasmids that expressed HA-NS2 and Flag-OTUB1."
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"Immunoblotting with anti-HA antibody revealed that HA-NS2 was coimmunoprecipitated with Flag-OTUB1 ( xref , lane 4)."
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"HA-NS2 was not coimmunoprecipitated by anti-Flag antibody if the cells were not transfected with pFlag-OTUB1 ( xref , lanes, 1, 3) or pHA-NS2 ( xref , lanes 1, 2)."
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"HEK293T cells were cotransfected with pHA-NS2, pV5-Ub, and 0.5 μg or 1 μg pFlag-OTUB1."
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"The level of polyubiquitinated HA-NS2 was reduced after the expression of Flag-OTUB1 in a dose-dependent manner ( xref , lanes 2–4) while the controls, α-tubulin, HA-NS2, and total ubiquitinated protein, in each lane were unaffected ( xref , lanes 5–8), indicating that the reduction of polyubiquitinated HA-NS2 was not due to sample loading variations or lack of HA-NS2 expression."
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"We found that although levels of NS2 conjugated with V5-Ub were substantially reduced if HEK293T cells were cotransfected with pFlag-OTUB1 ( xref , lanes 3, 4), no reduction of Ub-HA-NS2 was observed if cells were cotransfected with pFlag-mOTUB1 ( xref , lane 5), indicating that OTUB1 deubiquitinates NS2 through its catalytic activity."
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"We cotransfected HEK293T cells with pHA-NS2 and p3xFlag-myc-CMV26 or pHA-NS2 and pFlag-OTUB1 to determine whether the expression of OTUB1 stabilizes HA-NS2."
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"On the other hand, in cells cotransfected with pHA-NS2 and pFlag-OTUB1, HA-NS2 remained relatively stable, with a half-life of more than 90 min ( xref , lanes 5-8; xref )."
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