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USP12 binds luxY. 41 / 41
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"As shown in Fig. 3 c, the ability of YFP-USP12 delMEIL mutant to relocate to the PM when co-expressed with Myc-WDR20 was virtually abrogated."
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"In contrast, the YFP-USP12 ST/AA mutant still co-localized with Myc-WDR20 to the PM."
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"Importantly, co-IP analysis ( Fig. 3 d) showed that YFP-USP12 delMEIL interacts with Myc-WDR20 as efficiently as wild type YFP-USP12."
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"We generated YFP-USP12 V279D/F287A and Myc-WDR20 F262A/W306A mutants and confirmed that these mutations largely or completely abrogate WDR20 binding to USP12 in 293 T cells ( Fig. 4 a)."
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"As shown in Fig. 4 b, YFP-USP12 V279D/F287A did not relocate to the PM when co-expressed with wild type Myc-WDR20, and conversely, co-expression with Myc-WDR20 F262A/W306A did not result in PM localiz[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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"As in 293 T cells, YFP-USP12 also localized to the PM in live HeLa cells when co-expressed with Myc-WDR20 ( Fig. 4 c)."
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"The YFP-USP12 fluorescent signal was rapidly recovered in an area of the PM where it had been bleached."
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"The calculated halftime of recovery (t 1/2 ) and mobile fraction (F m ) values were 19.44 s and 65.29%, respectively, indicating that the PM localization of YFP-USP12 is highly dynamic."
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"The clearest and most statistically significant effect of LMB was on YFP-USP12 and, particularly, on YFP-WDR20, indicating that both proteins are actively exported from the nucleus by the CRM1-mediate[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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"Of note, YFP-USP12 and YFP-WDR20 were evenly distributed between nucleus and cytoplasm in LMB-treated cells, but they did not accumulate to a high level inside the nucleus."
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"To gauge these possibilities, we generated a version of YFP-USP12 bearing two copies of the strong SV40 large T antigen NLS (YFP-USP12 [+NLSs] ) ( Fig. 5 b)."
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"YFP-USP12 [+NLSs] readily accumulated into the nucleus, suggesting that YFP-USP12 inefficient import into the nucleus is most likely due to the lack of strong NLSs."
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"Of note, a faint fluorescent signal at the PM was also noticeable in some cells expressing YFP-USP12 [+NLSs] alone, probably due to the presence of endogenous WDR20."
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"The efficient nuclear import of YFP-USP12 [+NLSs] provided a convenient experimental tool to test our hypothesis that USP12/WDR20 may shuttle between the PM, cytoplasm and nucleus."
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"Despite the presence of the strong SV40 NLSs, YFP-USP12 [+NLSs] localized to the PM when co-expressed with Myc-WDR20 ( Fig. 5 c) in 293 T cells."
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"Importantly, YFP-USP12 [+NLSs] and Myc-WDR20 partially relocated to the nucleus when CRM1-mediated export was inhibited by LMB treatment."
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"In fact, live microscopy experiments revealed that YFP-USP12 [+NLSs] was detectable in the nucleus only a few minutes after LMB addition ( Fig. 5 d)."
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"We introduced these mutations into YFP-USP12 to generate YFP-USP12 “NES”m ."
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"The nucleocytoplasmic distribution of YFP-USP12 “NES”m was identical to that of YFP-USP12 in HeLa cells ( Fig. 6 c), further supporting our view that the motif 77 KESLLTCLADLFHSI 91 is not a direct de[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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"Using co-IP analyses, we found that YFP-USP12 “NES”m retained its ability to interact with Xpress-UAF1 ( Fig. 6 d, left)."
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"In order to test the possibility that WDR20 NES regulates the localization of USP12 deubiquitinase complexes, 293 T cells were co-transfected with YFP-USP12 [+NLSs] and either wild type or NES-mutant [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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"As shown in Fig. 8 a, YFP-USP12 [+NLSs] located almost exclusively to the PM when co-expressed with wild type Myc-WDR20."
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"In striking contrast, YFP-USP12 [+NLSs] located to both the nucleus and the PM when co-expressed with Myc-WDR20 NESm , a distribution that was similar to that of YFP-USP12 [+NLSs] co-expressed with wi[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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"To generate plasmids encoding YFP-USP12, YFP-USP12 V279D/F287A , YFP-USP12 ST/AA and YFP-WDR20, the corresponding cDNA sequences were purchased as gBlocks (IDT), and cloned into pEYFP-C1 (Clontech) us[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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"To generate YFP-USP12 [+NLSs] , USP12 cDNA was cloned into a modified version of pEYFP-C1 containing two copies of the SV40 large T antigen NLS."
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"On one hand, 293 T cells were co-transfected with YFP-USP12 [+NLSs] , UAF1-mRFP, and wild type Myc-WDR20 and either left untreated or treated with LMB."
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"On the other hand, cells were co-transfected with YFP-USP12 [+NLSs] , UAF1-mRFP and NES-mutant Myc-WDR20."
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"In line with previous studies ( Urbé et al., 2012 ; Lehoux et al., 2014 ), we found that YFP-USP12 and YFP-USP46 were located predominantly in the cytoplasm of 293 T and HeLa cells when expressed alon[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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"To generate YFP-USP12 delMEIL , YFP-USP46 +MEIL and YFP-WDR20 deletion mutants, the corresponding DNA sequences were amplified by PCR and cloned into pEYFP-C1 using BamHI/HindIII."
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"Finally, Myc-WDR20 F262A/W306A , Myc-WDR20 NESm , YFP-WDR20 NESm and YFP-USP12 “NES”m mutants were created using the Quick-Change Lightning Site-Directed Mutagenesis Kit (Stratagene)."
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"The most striking finding was the relocation of YFP-USP12, but not YFP-USP46 to the PM when co-expressed with Myc-WDR20."
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"In support of this view, we show that fusing two copies of the SV40 large T antigen NLS to YFP-USP12 readily induces its nuclear accumulation."
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"Remarkably, we found that the variant of USP12 carrying strong heterologous NLSs (YFP-USP12 [+NLSs] ) was exclusively located to the PM when co-expressed with Myc-WDR20, but partially relocated to the[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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"In order to asses a potential effect of UAF1 on the localization of YFP-USP12 and YFP-USP46, cellular levels of UAF1 were either reduced using small interfering RNA (siRNA)-mediated knockdown or incre[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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"This motif was non-functional in a nuclear export assay (Henderson and Eleftheriou, 2000), and mutations of this “NES” (unlike LMB treatment) did not decrease the cytoplasmic localization of YFP-USP12[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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"As shown in Fig. 1 b, UAF1 knockdown had no obvious effect on the localization of YFP-USP12 or YFP-USP46."
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"Of note, we used the YFP-USP12 [+NLSs] variant in these experiments as a tool to more clearly visualize the effect of WDR20 NES mutations."
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"On the other hand, YFP-USP12 and YFP-USP46 co-localized with UAF1-mRFP throughout the cytoplasm in co-transfection experiments ( Fig. 1 c) and, as shown by image analysis, co-expression with UAF1 mark[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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"We used a similar knockdown/overexpression approach to asses a potential effect of WDR20 on the localization of YFP-USP12 and YFP-USP46."
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"Similar to UAF1 knockdown, WDR20 siRNA had no obvious effect on the localization of YFP-USP12 or YFP-USP46 ( Fig. 2 a)."
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"Whereas YFP-USP46 and Myc-WDR20 co-localized diffusely throughout the cytoplasm, YFP-USP12 and Myc-WDR20 co-localized at the cell periphery ( Fig. 2 b)."
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