IndraLab

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OTUD3 binds MYC. 18 / 18
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"To confirm this interaction, we overexpressed OTUD3-Myc and KPTN-Flag in HEK-293T cells and performed immunoprecipitation experiments, which showed a clear interaction between the two proteins ( xref )."
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"To test this hypothesis, we transfected KPTN shRNA into PTEN-KO HeLa cells exogenously expressing OTUD3-Myc, observing whether OTUD3 could still inhibit the mTORC1 signaling pathway in the absence of both PTEN and KPTN."
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"Thus, to explore this issue we transiently transfected wild-type HeLa cells with OTUD3-Myc and catalytically inactive OTUD3C76A-Myc, using HeLa cells transfected with an empty vector as a control."
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"In subsequent experiments, we exogenously expressed OTUD3-Myc in PTEN-knockout HeLa cells and subjected these cells to time-gradient amino acid starvation or amino acid starvation followed by stimulation."
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"Additionally, we transfected OTUD3-Myc as an experimental group in HeLa cells with endogenous OTUD3 knocked out or HeLa cells with endogenous KPTN knocked out."
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"We reintroduced exogenous OTUD3-Myc and catalytically inactive OTUD3C76A-Myc into these cells and subjected them to a time gradient of amino acid starvation or amino acid stimulation after starvation."
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"We performed co-immunoprecipitation with KPTN-Flag using OTUD3-Myc and OTUD3C76A-Myc and found that the enzymatically inactive OTUD3C76A retains its ability to interact with KPTN-Flag ( xref )."
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"Overexpressed KPTN-Flag and Ub-HA HEK-293T cells, along with varying levels of OTUD3-Myc or enzymatically inactive OTUD3C76A-Myc, were established."
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"Myc-OTUD3 and Flag-SMAD1-7 expressing vectors were co-transfected into HEK293T cells, and the cell lysate was subjected to co-immunoprecipitation."
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"Interestingly, in wild-type HeLa cells overexpressing OTUD3-Myc, although the phosphorylation trend of S6K1 was similar, the overall phosphorylation level was significantly reduced compared to wild-type HeLa cells ( xref )."
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"Our study revealed that in HeLa cells expressing either wild-type OTUD3-Myc or catalytically inactive OTUD3C76A-Myc, the phosphorylation trend of S6K1 was similar."
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"This might be due to the transient overexpression of exogenous OTUD3-Myc leading to significantly higher protein levels than the stable overexpression of endogenous OTUD3, resulting in higher suppression of the mTORC1 signaling pathway and influencing HeLa cell proliferation rates."
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"However, when compared to HeLa cells overexpressing wild-type OTUD3-Myc, HeLa cells overexpressing catalytically inactive OTUD3C76A-Myc exhibited an overall increase in S6K1 phosphorylation levels, which were closer to those of the wild-type HeLa control group ( xref )."
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"This finding indicates that the upregulation of the mTORC1 signaling pathway caused by PTEN-KO cells can be suppressed by exogenous expression of OTUD3-Myc."
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"In KPTN-knockout HeLa cells, we exogenously transfected OTUD3-Myc and empty vector, subjecting these cells to time-gradient amino acid starvation or amino acid starvation followed by stimulation."
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"Moreover, during amino acid starvation, HeLa cells overexpressing wild-type OTUD3-Myc showed a faster decrease in S6K1 phosphorylation levels, while during amino acid stimulation after starvation, the increase was slower."
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"The results indicated that in KPTN-knockout HeLa cells, exogenously expressed OTUD3-Myc did not exhibit the same ability to significantly reduce the overall phosphorylation level of S6K1 as shown previously, and the levels were comparable to those of KPTN-knockout cells transfected with the empty vector ( xref )."
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"To determine whether the OTUD3-TP53 interaction was direct, we generated and purified recombinant Myc-OTUD3, Myc-D1 and Myc-D2."
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