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USP4 binds BRCA1. 20 / 20
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"Based on this, we then examined whether USP4 and BRCA1 could interact with each other in the absence of CtIP."
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"To investigate the physiological significance of USP4-BRCA1 association, we analyzed the protein level and interaction between BRCA1 and USP4 during cell cycle."
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"Immunoprecipitation result showed that the interaction between USP4 and BRCA1 is almost undetectable at early G1, and increases from late G1 through S, and peaks at G2/M, which concomitantly correlates with the levels of BRCA1 at these phases (Fig. xref )."
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"This result is consistent with a role of USP4 in stabilizing BRCA1, suggesting that USP4 physiologically interacts with BRCA1 to control BRCA1 protein level during the progression of cell cycle."
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"The Co-IP assay indicated that endogenous BRCA1 could interact with USP4 in a manner that is independent of its DUB activity (Supplementary Fig. xref )."
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"This result indicates that USP4 can interact with both BARD1-unbound BRCA1(“free” BRCA1) and may also bind BRCA1/BARD1 heterodimer."
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"This result suggests that USP4 can interact with BRCA1 in a manner independent of CtIP."
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"Interestingly, the interaction between USP4 and BRCA1 was enhanced with IR/ doxorubicin treatment (Fig. xref ), suggesting their interactions might be DNA damage inducible."
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"To further confirm the in vivo interaction between USP4 and BRCA1, Co-IP of endogenous USP4 and BRCA1 from MCF-10A cells were performed and the results showed that USP4 was efficiently co-immunoprecipitated with BRCA1, and vice versa (Fig. xref )."
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"USP4 physically interacts with and deubiquitinates BRCA1, thus saving BRCA1 from UPS-meditated ubiquitination and degradation."
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"To further confirm the in vivo interaction between USP4 and BRCA1, Co-IP of endogenous USP4 and BRCA1 from MCF-10A cells were performed and the results showed that USP4 was efficiently co-immunoprecipitated with BRCA1, and vice versa (Fig. 2b, c)."
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"Interestingly, the interaction between USP4 and BRCA1 was enhanced with IR/ doxorubicin treatment (Fig. 2d), suggesting their interactions might be DNA damage inducible.BRCA1 protein fluctuates during cell cycle, with its level low at G1, increases from G1 through S phase, and peaks at G2/M phase."
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"Immunoprecipitation result showed that the interaction between USP4 and BRCA1 is almost undetectable at early G1, and increases from late G1 through S, and peaks at G2/M, which concomitantly correlates with the levels of BRCA1 at these phases (Fig. 2e)."
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"This result is consistent with a role of USP4 in stabilizing BRCA1, suggesting that USP4 physiologically interacts with BRCA1 to control BRCA1 protein level during the progression of cell cycle.To confirm the interaction between USP4 and BRCA1, we performed binding assay using recombinant USP4 fused with GST (GST-USP4)."
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"To confirm the interaction of USP4 with BRCA1, HEK293T cells were transfected with Flag-tagged USP4 WT or the C311A mutant plasmid and a Co-IP experiment was performed."
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"To verify the functional association between USP4 and BRCA1 in this process, we reintroduced BRCA1 in MDA-MB-231 cells stably depleted of USP4, and cells were then exposed to doxorubicin treatment."
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"To confirm the interaction between USP4 and BRCA1, we performed binding assay using recombinant USP4 fused with GST (GST-USP4)."
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"The results showed that the purified GST-USP4 but not the GST control was able to pull-down BRCA1 in vitro (Fig. xref ), suggesting that a direct interaction might exist between USP4 and BRCA1."
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"Similar results were obtained when assessing ubiquitination of other BRCA1 truncations such as F2 and F3 (Fig. xref , Supplementary Fig. xref ), which dominated the interaction between USP4 and BRCA1."
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"Notably, this USP4-mediated de-ubiquitination of BRCA1 is CtIP independent, indicating that USP4-BRCA1 axis is a distinct pathway other than USP4-CtIP in regulating HR."
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