prs415 expression vector Search Results


gfp n180 ubp3 3 myc  (ATCC)
94
ATCC gfp n180 ubp3 3 myc
Yeast strains used in this study
Gfp N180 Ubp3 3 Myc, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/gfp n180 ubp3 3 myc/product/ATCC
Average 94 stars, based on 1 article reviews
gfp n180 ubp3 3 myc - by Bioz Stars, 2026-03
94/100 stars
  Buy from Supplier
spei  (New England Biolabs)
99
New England Biolabs spei
Yeast strains used in this study
Spei, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/spei/product/New England Biolabs
Average 99 stars, based on 1 article reviews
spei - by Bioz Stars, 2026-03
99/100 stars
  Buy from Supplier
yeast expression plasmid prs425  (Addgene inc)
90
Addgene inc yeast expression plasmid prs425
Yeast strains used in this study
Yeast Expression Plasmid Prs425, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/yeast expression plasmid prs425/product/Addgene inc
Average 90 stars, based on 1 article reviews
yeast expression plasmid prs425 - by Bioz Stars, 2026-03
90/100 stars
  Buy from Supplier
miniturbo tag  (Addgene inc)
93
Addgene inc miniturbo tag
( A ) Schematic overview of the CRISPR screen for identifying DUBs regulating Itgb1 surface levels. Cas9-expressing HAP1 cells were transduced with pooled lentiviral guide RNA (gRNA) libraries targeting 98 DUBs from the human genome. After 2 weeks in culture, cells with the 5% lowest (Itgb1 Lo ) and the 5% highest (Itgb1 Hi ) Itgb1 surface levels were sorted by flow cytometry, and gRNA-targeted genes were determined. ( B ) Volcano plot of the results from the CRISPR screen. The x-axis represents the log 2 fold change (lfc) in the frequency of genes targeted between the Itgb1 Lo and Itgb1 Hi populations. The y-axis indicates the robust rank aggregation (RRA) score determined by the MAGeCK algorithm (MAGeCK-RRA) (Li et al, ). Dots represent individual targeted genes, and those meeting the criteria of |lfc| >0.33 and −log 10 (RRA) >2 were considered significant. Genes significantly enriched in Itgb1 Lo cells are colored in blue, and those enriched in Itgb1 Hi in red. USP12 is marked in white. ( C ) Volcano plot of the α5β1 integrin proximitome determined by label-free MS analysis in mouse kidney fibroblasts expressing <t>miniTurbo-tagged</t> Itag5 (TurboID) versus Itgb1-KO fibroblasts (Ctrl). P values were determined using a two-sided permuted t -test with 250 randomizations. The black dashed line indicates the significance cutoff (FDR:0.05, S0:0.1) estimated by the Perseus software. n = 3 biological replicates. The red dots indicate the subunits of the α5β1 heterodimer and the components of the USP12/46-WDR48-WDR20 complex. ( D ) Itgb1 surface levels in WT and two independent clones (cl1 and cl2) of USP12-KO, USP46-KO, and USP12/46-dKO fibroblasts determined by flow cytometry. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test comparing the WT fibroblasts with USP12-KO cl1 or cl2, USP46-KO cl1 or cl2, USP12/46-KO cl1 or cl2 fibroblasts ( P = 0.9905, 0.9968, 0.2401, 0.8080, 0.0067, and 0.0065, respectively). ** P < 0.01; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( E , F ) WB ( E ) and densitometric quantification ( F ) of Itgb1 and Itga5 protein levels in WT and USP12/46-dKO fibroblasts. Gapdh served as loading control. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test comparing the WT fibroblasts with USP12/46-KO cl1 or cl2 fibroblasts (for Itgb1, P = 0.0162 and 0.0189, respectively; for Itga5, P = 0.0180 and 0.0106, respectively). * P < 0.05. Data were shown as Mean ± SD, n = 3 independent experiments. ( G – I ) Itgb1 surface levels were determined by flow cytometry ( G ), Itgb1 protein levels in cell lysates were determined by WB ( H ), and densitometric quantification ( I ) in WT and USP12/46-dKO fibroblasts stably expressing EGFP, EGFP-USP12 WT , or EGFP-USP12 C48S . Gapdh served as a loading control. Statistical analysis was carried out by RM two-way ANOVA with Dunnett’s multiple comparison test. In ( G ), statistical significance was tested comparing the EGFP group with EGFP-USP12 WT or EGFP-USP12 C48S group in WT fibroblasts ( P = 0.3449 and 0.0193, respectively); in USP12/46-dKO cl1 fibroblasts ( P = 0.0335 and 0.6230, respectively); and in USP12/46-dKO cl2 fibroblasts ( P = 0.0184 and 0.9260, respectively). In ( I ), statistical significance was tested comparing the EGFP group with EGFP-USP12 WT or EGFP-USP12 C48S group in WT fibroblasts ( P = 0.6467 and 0.6716, respectively); in USP12/46-dKO cl1 fibroblasts ( P = 0.0266 and 0.3351, respectively); and in USP12/46-dKO cl2 fibroblasts ( P = 0.0332 and 0.1310, respectively). * P < 0.05; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( J ) Volcano plot of the cell surface proteome of USP12/46-dKO fibroblasts stably expressing EGFP-USP12 C48S versus EGFP-USP12 WT identified by label-free MS. P values were determined using two-sided permuted t -test with 250 randomizations. The black dashed line indicates the significance cutoff (FDR:0.05, S0:0.1) estimated by the Perseus software. n = 4 biological replicates. Arbitrarily selected cell surface receptors were highlighted in red. .
Miniturbo Tag, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/miniturbo tag/product/Addgene inc
Average 93 stars, based on 1 article reviews
miniturbo tag - by Bioz Stars, 2026-03
93/100 stars
  Buy from Supplier
prs415 expression vector  (Addgene inc)
91
Addgene inc prs415 expression vector
( A ) Schematic overview of the CRISPR screen for identifying DUBs regulating Itgb1 surface levels. Cas9-expressing HAP1 cells were transduced with pooled lentiviral guide RNA (gRNA) libraries targeting 98 DUBs from the human genome. After 2 weeks in culture, cells with the 5% lowest (Itgb1 Lo ) and the 5% highest (Itgb1 Hi ) Itgb1 surface levels were sorted by flow cytometry, and gRNA-targeted genes were determined. ( B ) Volcano plot of the results from the CRISPR screen. The x-axis represents the log 2 fold change (lfc) in the frequency of genes targeted between the Itgb1 Lo and Itgb1 Hi populations. The y-axis indicates the robust rank aggregation (RRA) score determined by the MAGeCK algorithm (MAGeCK-RRA) (Li et al, ). Dots represent individual targeted genes, and those meeting the criteria of |lfc| >0.33 and −log 10 (RRA) >2 were considered significant. Genes significantly enriched in Itgb1 Lo cells are colored in blue, and those enriched in Itgb1 Hi in red. USP12 is marked in white. ( C ) Volcano plot of the α5β1 integrin proximitome determined by label-free MS analysis in mouse kidney fibroblasts expressing <t>miniTurbo-tagged</t> Itag5 (TurboID) versus Itgb1-KO fibroblasts (Ctrl). P values were determined using a two-sided permuted t -test with 250 randomizations. The black dashed line indicates the significance cutoff (FDR:0.05, S0:0.1) estimated by the Perseus software. n = 3 biological replicates. The red dots indicate the subunits of the α5β1 heterodimer and the components of the USP12/46-WDR48-WDR20 complex. ( D ) Itgb1 surface levels in WT and two independent clones (cl1 and cl2) of USP12-KO, USP46-KO, and USP12/46-dKO fibroblasts determined by flow cytometry. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test comparing the WT fibroblasts with USP12-KO cl1 or cl2, USP46-KO cl1 or cl2, USP12/46-KO cl1 or cl2 fibroblasts ( P = 0.9905, 0.9968, 0.2401, 0.8080, 0.0067, and 0.0065, respectively). ** P < 0.01; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( E , F ) WB ( E ) and densitometric quantification ( F ) of Itgb1 and Itga5 protein levels in WT and USP12/46-dKO fibroblasts. Gapdh served as loading control. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test comparing the WT fibroblasts with USP12/46-KO cl1 or cl2 fibroblasts (for Itgb1, P = 0.0162 and 0.0189, respectively; for Itga5, P = 0.0180 and 0.0106, respectively). * P < 0.05. Data were shown as Mean ± SD, n = 3 independent experiments. ( G – I ) Itgb1 surface levels were determined by flow cytometry ( G ), Itgb1 protein levels in cell lysates were determined by WB ( H ), and densitometric quantification ( I ) in WT and USP12/46-dKO fibroblasts stably expressing EGFP, EGFP-USP12 WT , or EGFP-USP12 C48S . Gapdh served as a loading control. Statistical analysis was carried out by RM two-way ANOVA with Dunnett’s multiple comparison test. In ( G ), statistical significance was tested comparing the EGFP group with EGFP-USP12 WT or EGFP-USP12 C48S group in WT fibroblasts ( P = 0.3449 and 0.0193, respectively); in USP12/46-dKO cl1 fibroblasts ( P = 0.0335 and 0.6230, respectively); and in USP12/46-dKO cl2 fibroblasts ( P = 0.0184 and 0.9260, respectively). In ( I ), statistical significance was tested comparing the EGFP group with EGFP-USP12 WT or EGFP-USP12 C48S group in WT fibroblasts ( P = 0.6467 and 0.6716, respectively); in USP12/46-dKO cl1 fibroblasts ( P = 0.0266 and 0.3351, respectively); and in USP12/46-dKO cl2 fibroblasts ( P = 0.0332 and 0.1310, respectively). * P < 0.05; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( J ) Volcano plot of the cell surface proteome of USP12/46-dKO fibroblasts stably expressing EGFP-USP12 C48S versus EGFP-USP12 WT identified by label-free MS. P values were determined using two-sided permuted t -test with 250 randomizations. The black dashed line indicates the significance cutoff (FDR:0.05, S0:0.1) estimated by the Perseus software. n = 4 biological replicates. Arbitrarily selected cell surface receptors were highlighted in red. .
Prs415 Expression Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/prs415 expression vector/product/Addgene inc
Average 91 stars, based on 1 article reviews
prs415 expression vector - by Bioz Stars, 2026-03
91/100 stars
  Buy from Supplier
pme18sfl3  (Toyobo)
90
Toyobo pme18sfl3
( A ) Schematic overview of the CRISPR screen for identifying DUBs regulating Itgb1 surface levels. Cas9-expressing HAP1 cells were transduced with pooled lentiviral guide RNA (gRNA) libraries targeting 98 DUBs from the human genome. After 2 weeks in culture, cells with the 5% lowest (Itgb1 Lo ) and the 5% highest (Itgb1 Hi ) Itgb1 surface levels were sorted by flow cytometry, and gRNA-targeted genes were determined. ( B ) Volcano plot of the results from the CRISPR screen. The x-axis represents the log 2 fold change (lfc) in the frequency of genes targeted between the Itgb1 Lo and Itgb1 Hi populations. The y-axis indicates the robust rank aggregation (RRA) score determined by the MAGeCK algorithm (MAGeCK-RRA) (Li et al, ). Dots represent individual targeted genes, and those meeting the criteria of |lfc| >0.33 and −log 10 (RRA) >2 were considered significant. Genes significantly enriched in Itgb1 Lo cells are colored in blue, and those enriched in Itgb1 Hi in red. USP12 is marked in white. ( C ) Volcano plot of the α5β1 integrin proximitome determined by label-free MS analysis in mouse kidney fibroblasts expressing <t>miniTurbo-tagged</t> Itag5 (TurboID) versus Itgb1-KO fibroblasts (Ctrl). P values were determined using a two-sided permuted t -test with 250 randomizations. The black dashed line indicates the significance cutoff (FDR:0.05, S0:0.1) estimated by the Perseus software. n = 3 biological replicates. The red dots indicate the subunits of the α5β1 heterodimer and the components of the USP12/46-WDR48-WDR20 complex. ( D ) Itgb1 surface levels in WT and two independent clones (cl1 and cl2) of USP12-KO, USP46-KO, and USP12/46-dKO fibroblasts determined by flow cytometry. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test comparing the WT fibroblasts with USP12-KO cl1 or cl2, USP46-KO cl1 or cl2, USP12/46-KO cl1 or cl2 fibroblasts ( P = 0.9905, 0.9968, 0.2401, 0.8080, 0.0067, and 0.0065, respectively). ** P < 0.01; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( E , F ) WB ( E ) and densitometric quantification ( F ) of Itgb1 and Itga5 protein levels in WT and USP12/46-dKO fibroblasts. Gapdh served as loading control. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test comparing the WT fibroblasts with USP12/46-KO cl1 or cl2 fibroblasts (for Itgb1, P = 0.0162 and 0.0189, respectively; for Itga5, P = 0.0180 and 0.0106, respectively). * P < 0.05. Data were shown as Mean ± SD, n = 3 independent experiments. ( G – I ) Itgb1 surface levels were determined by flow cytometry ( G ), Itgb1 protein levels in cell lysates were determined by WB ( H ), and densitometric quantification ( I ) in WT and USP12/46-dKO fibroblasts stably expressing EGFP, EGFP-USP12 WT , or EGFP-USP12 C48S . Gapdh served as a loading control. Statistical analysis was carried out by RM two-way ANOVA with Dunnett’s multiple comparison test. In ( G ), statistical significance was tested comparing the EGFP group with EGFP-USP12 WT or EGFP-USP12 C48S group in WT fibroblasts ( P = 0.3449 and 0.0193, respectively); in USP12/46-dKO cl1 fibroblasts ( P = 0.0335 and 0.6230, respectively); and in USP12/46-dKO cl2 fibroblasts ( P = 0.0184 and 0.9260, respectively). In ( I ), statistical significance was tested comparing the EGFP group with EGFP-USP12 WT or EGFP-USP12 C48S group in WT fibroblasts ( P = 0.6467 and 0.6716, respectively); in USP12/46-dKO cl1 fibroblasts ( P = 0.0266 and 0.3351, respectively); and in USP12/46-dKO cl2 fibroblasts ( P = 0.0332 and 0.1310, respectively). * P < 0.05; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( J ) Volcano plot of the cell surface proteome of USP12/46-dKO fibroblasts stably expressing EGFP-USP12 C48S versus EGFP-USP12 WT identified by label-free MS. P values were determined using two-sided permuted t -test with 250 randomizations. The black dashed line indicates the significance cutoff (FDR:0.05, S0:0.1) estimated by the Perseus software. n = 4 biological replicates. Arbitrarily selected cell surface receptors were highlighted in red. .
Pme18sfl3, supplied by Toyobo, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pme18sfl3/product/Toyobo
Average 90 stars, based on 1 article reviews
pme18sfl3 - by Bioz Stars, 2026-03
90/100 stars
  Buy from Supplier
pqe-80l  (Qiagen)
90
Qiagen pqe-80l
( A ) Schematic overview of the CRISPR screen for identifying DUBs regulating Itgb1 surface levels. Cas9-expressing HAP1 cells were transduced with pooled lentiviral guide RNA (gRNA) libraries targeting 98 DUBs from the human genome. After 2 weeks in culture, cells with the 5% lowest (Itgb1 Lo ) and the 5% highest (Itgb1 Hi ) Itgb1 surface levels were sorted by flow cytometry, and gRNA-targeted genes were determined. ( B ) Volcano plot of the results from the CRISPR screen. The x-axis represents the log 2 fold change (lfc) in the frequency of genes targeted between the Itgb1 Lo and Itgb1 Hi populations. The y-axis indicates the robust rank aggregation (RRA) score determined by the MAGeCK algorithm (MAGeCK-RRA) (Li et al, ). Dots represent individual targeted genes, and those meeting the criteria of |lfc| >0.33 and −log 10 (RRA) >2 were considered significant. Genes significantly enriched in Itgb1 Lo cells are colored in blue, and those enriched in Itgb1 Hi in red. USP12 is marked in white. ( C ) Volcano plot of the α5β1 integrin proximitome determined by label-free MS analysis in mouse kidney fibroblasts expressing <t>miniTurbo-tagged</t> Itag5 (TurboID) versus Itgb1-KO fibroblasts (Ctrl). P values were determined using a two-sided permuted t -test with 250 randomizations. The black dashed line indicates the significance cutoff (FDR:0.05, S0:0.1) estimated by the Perseus software. n = 3 biological replicates. The red dots indicate the subunits of the α5β1 heterodimer and the components of the USP12/46-WDR48-WDR20 complex. ( D ) Itgb1 surface levels in WT and two independent clones (cl1 and cl2) of USP12-KO, USP46-KO, and USP12/46-dKO fibroblasts determined by flow cytometry. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test comparing the WT fibroblasts with USP12-KO cl1 or cl2, USP46-KO cl1 or cl2, USP12/46-KO cl1 or cl2 fibroblasts ( P = 0.9905, 0.9968, 0.2401, 0.8080, 0.0067, and 0.0065, respectively). ** P < 0.01; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( E , F ) WB ( E ) and densitometric quantification ( F ) of Itgb1 and Itga5 protein levels in WT and USP12/46-dKO fibroblasts. Gapdh served as loading control. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test comparing the WT fibroblasts with USP12/46-KO cl1 or cl2 fibroblasts (for Itgb1, P = 0.0162 and 0.0189, respectively; for Itga5, P = 0.0180 and 0.0106, respectively). * P < 0.05. Data were shown as Mean ± SD, n = 3 independent experiments. ( G – I ) Itgb1 surface levels were determined by flow cytometry ( G ), Itgb1 protein levels in cell lysates were determined by WB ( H ), and densitometric quantification ( I ) in WT and USP12/46-dKO fibroblasts stably expressing EGFP, EGFP-USP12 WT , or EGFP-USP12 C48S . Gapdh served as a loading control. Statistical analysis was carried out by RM two-way ANOVA with Dunnett’s multiple comparison test. In ( G ), statistical significance was tested comparing the EGFP group with EGFP-USP12 WT or EGFP-USP12 C48S group in WT fibroblasts ( P = 0.3449 and 0.0193, respectively); in USP12/46-dKO cl1 fibroblasts ( P = 0.0335 and 0.6230, respectively); and in USP12/46-dKO cl2 fibroblasts ( P = 0.0184 and 0.9260, respectively). In ( I ), statistical significance was tested comparing the EGFP group with EGFP-USP12 WT or EGFP-USP12 C48S group in WT fibroblasts ( P = 0.6467 and 0.6716, respectively); in USP12/46-dKO cl1 fibroblasts ( P = 0.0266 and 0.3351, respectively); and in USP12/46-dKO cl2 fibroblasts ( P = 0.0332 and 0.1310, respectively). * P < 0.05; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( J ) Volcano plot of the cell surface proteome of USP12/46-dKO fibroblasts stably expressing EGFP-USP12 C48S versus EGFP-USP12 WT identified by label-free MS. P values were determined using two-sided permuted t -test with 250 randomizations. The black dashed line indicates the significance cutoff (FDR:0.05, S0:0.1) estimated by the Perseus software. n = 4 biological replicates. Arbitrarily selected cell surface receptors were highlighted in red. .
Pqe 80l, supplied by Qiagen, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pqe-80l/product/Qiagen
Average 90 stars, based on 1 article reviews
pqe-80l - by Bioz Stars, 2026-03
90/100 stars
  Buy from Supplier
prs415  (Agilent technologies)
90
Agilent technologies prs415
( A ) Schematic overview of the CRISPR screen for identifying DUBs regulating Itgb1 surface levels. Cas9-expressing HAP1 cells were transduced with pooled lentiviral guide RNA (gRNA) libraries targeting 98 DUBs from the human genome. After 2 weeks in culture, cells with the 5% lowest (Itgb1 Lo ) and the 5% highest (Itgb1 Hi ) Itgb1 surface levels were sorted by flow cytometry, and gRNA-targeted genes were determined. ( B ) Volcano plot of the results from the CRISPR screen. The x-axis represents the log 2 fold change (lfc) in the frequency of genes targeted between the Itgb1 Lo and Itgb1 Hi populations. The y-axis indicates the robust rank aggregation (RRA) score determined by the MAGeCK algorithm (MAGeCK-RRA) (Li et al, ). Dots represent individual targeted genes, and those meeting the criteria of |lfc| >0.33 and −log 10 (RRA) >2 were considered significant. Genes significantly enriched in Itgb1 Lo cells are colored in blue, and those enriched in Itgb1 Hi in red. USP12 is marked in white. ( C ) Volcano plot of the α5β1 integrin proximitome determined by label-free MS analysis in mouse kidney fibroblasts expressing <t>miniTurbo-tagged</t> Itag5 (TurboID) versus Itgb1-KO fibroblasts (Ctrl). P values were determined using a two-sided permuted t -test with 250 randomizations. The black dashed line indicates the significance cutoff (FDR:0.05, S0:0.1) estimated by the Perseus software. n = 3 biological replicates. The red dots indicate the subunits of the α5β1 heterodimer and the components of the USP12/46-WDR48-WDR20 complex. ( D ) Itgb1 surface levels in WT and two independent clones (cl1 and cl2) of USP12-KO, USP46-KO, and USP12/46-dKO fibroblasts determined by flow cytometry. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test comparing the WT fibroblasts with USP12-KO cl1 or cl2, USP46-KO cl1 or cl2, USP12/46-KO cl1 or cl2 fibroblasts ( P = 0.9905, 0.9968, 0.2401, 0.8080, 0.0067, and 0.0065, respectively). ** P < 0.01; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( E , F ) WB ( E ) and densitometric quantification ( F ) of Itgb1 and Itga5 protein levels in WT and USP12/46-dKO fibroblasts. Gapdh served as loading control. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test comparing the WT fibroblasts with USP12/46-KO cl1 or cl2 fibroblasts (for Itgb1, P = 0.0162 and 0.0189, respectively; for Itga5, P = 0.0180 and 0.0106, respectively). * P < 0.05. Data were shown as Mean ± SD, n = 3 independent experiments. ( G – I ) Itgb1 surface levels were determined by flow cytometry ( G ), Itgb1 protein levels in cell lysates were determined by WB ( H ), and densitometric quantification ( I ) in WT and USP12/46-dKO fibroblasts stably expressing EGFP, EGFP-USP12 WT , or EGFP-USP12 C48S . Gapdh served as a loading control. Statistical analysis was carried out by RM two-way ANOVA with Dunnett’s multiple comparison test. In ( G ), statistical significance was tested comparing the EGFP group with EGFP-USP12 WT or EGFP-USP12 C48S group in WT fibroblasts ( P = 0.3449 and 0.0193, respectively); in USP12/46-dKO cl1 fibroblasts ( P = 0.0335 and 0.6230, respectively); and in USP12/46-dKO cl2 fibroblasts ( P = 0.0184 and 0.9260, respectively). In ( I ), statistical significance was tested comparing the EGFP group with EGFP-USP12 WT or EGFP-USP12 C48S group in WT fibroblasts ( P = 0.6467 and 0.6716, respectively); in USP12/46-dKO cl1 fibroblasts ( P = 0.0266 and 0.3351, respectively); and in USP12/46-dKO cl2 fibroblasts ( P = 0.0332 and 0.1310, respectively). * P < 0.05; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( J ) Volcano plot of the cell surface proteome of USP12/46-dKO fibroblasts stably expressing EGFP-USP12 C48S versus EGFP-USP12 WT identified by label-free MS. P values were determined using two-sided permuted t -test with 250 randomizations. The black dashed line indicates the significance cutoff (FDR:0.05, S0:0.1) estimated by the Perseus software. n = 4 biological replicates. Arbitrarily selected cell surface receptors were highlighted in red. .
Prs415, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/prs415/product/Agilent technologies
Average 90 stars, based on 1 article reviews
prs415 - by Bioz Stars, 2026-03
90/100 stars
  Buy from Supplier
spha cassette  (Thermo Fisher)
90
Thermo Fisher spha cassette
( A ) Schematic overview of the CRISPR screen for identifying DUBs regulating Itgb1 surface levels. Cas9-expressing HAP1 cells were transduced with pooled lentiviral guide RNA (gRNA) libraries targeting 98 DUBs from the human genome. After 2 weeks in culture, cells with the 5% lowest (Itgb1 Lo ) and the 5% highest (Itgb1 Hi ) Itgb1 surface levels were sorted by flow cytometry, and gRNA-targeted genes were determined. ( B ) Volcano plot of the results from the CRISPR screen. The x-axis represents the log 2 fold change (lfc) in the frequency of genes targeted between the Itgb1 Lo and Itgb1 Hi populations. The y-axis indicates the robust rank aggregation (RRA) score determined by the MAGeCK algorithm (MAGeCK-RRA) (Li et al, ). Dots represent individual targeted genes, and those meeting the criteria of |lfc| >0.33 and −log 10 (RRA) >2 were considered significant. Genes significantly enriched in Itgb1 Lo cells are colored in blue, and those enriched in Itgb1 Hi in red. USP12 is marked in white. ( C ) Volcano plot of the α5β1 integrin proximitome determined by label-free MS analysis in mouse kidney fibroblasts expressing <t>miniTurbo-tagged</t> Itag5 (TurboID) versus Itgb1-KO fibroblasts (Ctrl). P values were determined using a two-sided permuted t -test with 250 randomizations. The black dashed line indicates the significance cutoff (FDR:0.05, S0:0.1) estimated by the Perseus software. n = 3 biological replicates. The red dots indicate the subunits of the α5β1 heterodimer and the components of the USP12/46-WDR48-WDR20 complex. ( D ) Itgb1 surface levels in WT and two independent clones (cl1 and cl2) of USP12-KO, USP46-KO, and USP12/46-dKO fibroblasts determined by flow cytometry. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test comparing the WT fibroblasts with USP12-KO cl1 or cl2, USP46-KO cl1 or cl2, USP12/46-KO cl1 or cl2 fibroblasts ( P = 0.9905, 0.9968, 0.2401, 0.8080, 0.0067, and 0.0065, respectively). ** P < 0.01; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( E , F ) WB ( E ) and densitometric quantification ( F ) of Itgb1 and Itga5 protein levels in WT and USP12/46-dKO fibroblasts. Gapdh served as loading control. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test comparing the WT fibroblasts with USP12/46-KO cl1 or cl2 fibroblasts (for Itgb1, P = 0.0162 and 0.0189, respectively; for Itga5, P = 0.0180 and 0.0106, respectively). * P < 0.05. Data were shown as Mean ± SD, n = 3 independent experiments. ( G – I ) Itgb1 surface levels were determined by flow cytometry ( G ), Itgb1 protein levels in cell lysates were determined by WB ( H ), and densitometric quantification ( I ) in WT and USP12/46-dKO fibroblasts stably expressing EGFP, EGFP-USP12 WT , or EGFP-USP12 C48S . Gapdh served as a loading control. Statistical analysis was carried out by RM two-way ANOVA with Dunnett’s multiple comparison test. In ( G ), statistical significance was tested comparing the EGFP group with EGFP-USP12 WT or EGFP-USP12 C48S group in WT fibroblasts ( P = 0.3449 and 0.0193, respectively); in USP12/46-dKO cl1 fibroblasts ( P = 0.0335 and 0.6230, respectively); and in USP12/46-dKO cl2 fibroblasts ( P = 0.0184 and 0.9260, respectively). In ( I ), statistical significance was tested comparing the EGFP group with EGFP-USP12 WT or EGFP-USP12 C48S group in WT fibroblasts ( P = 0.6467 and 0.6716, respectively); in USP12/46-dKO cl1 fibroblasts ( P = 0.0266 and 0.3351, respectively); and in USP12/46-dKO cl2 fibroblasts ( P = 0.0332 and 0.1310, respectively). * P < 0.05; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( J ) Volcano plot of the cell surface proteome of USP12/46-dKO fibroblasts stably expressing EGFP-USP12 C48S versus EGFP-USP12 WT identified by label-free MS. P values were determined using two-sided permuted t -test with 250 randomizations. The black dashed line indicates the significance cutoff (FDR:0.05, S0:0.1) estimated by the Perseus software. n = 4 biological replicates. Arbitrarily selected cell surface receptors were highlighted in red. .
Spha Cassette, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/spha cassette/product/Thermo Fisher
Average 90 stars, based on 1 article reviews
spha cassette - by Bioz Stars, 2026-03
90/100 stars
  Buy from Supplier
pacyc184 pa15a origin replication  (Millipore)
90
Millipore pacyc184 pa15a origin replication
( A ) Schematic overview of the CRISPR screen for identifying DUBs regulating Itgb1 surface levels. Cas9-expressing HAP1 cells were transduced with pooled lentiviral guide RNA (gRNA) libraries targeting 98 DUBs from the human genome. After 2 weeks in culture, cells with the 5% lowest (Itgb1 Lo ) and the 5% highest (Itgb1 Hi ) Itgb1 surface levels were sorted by flow cytometry, and gRNA-targeted genes were determined. ( B ) Volcano plot of the results from the CRISPR screen. The x-axis represents the log 2 fold change (lfc) in the frequency of genes targeted between the Itgb1 Lo and Itgb1 Hi populations. The y-axis indicates the robust rank aggregation (RRA) score determined by the MAGeCK algorithm (MAGeCK-RRA) (Li et al, ). Dots represent individual targeted genes, and those meeting the criteria of |lfc| >0.33 and −log 10 (RRA) >2 were considered significant. Genes significantly enriched in Itgb1 Lo cells are colored in blue, and those enriched in Itgb1 Hi in red. USP12 is marked in white. ( C ) Volcano plot of the α5β1 integrin proximitome determined by label-free MS analysis in mouse kidney fibroblasts expressing <t>miniTurbo-tagged</t> Itag5 (TurboID) versus Itgb1-KO fibroblasts (Ctrl). P values were determined using a two-sided permuted t -test with 250 randomizations. The black dashed line indicates the significance cutoff (FDR:0.05, S0:0.1) estimated by the Perseus software. n = 3 biological replicates. The red dots indicate the subunits of the α5β1 heterodimer and the components of the USP12/46-WDR48-WDR20 complex. ( D ) Itgb1 surface levels in WT and two independent clones (cl1 and cl2) of USP12-KO, USP46-KO, and USP12/46-dKO fibroblasts determined by flow cytometry. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test comparing the WT fibroblasts with USP12-KO cl1 or cl2, USP46-KO cl1 or cl2, USP12/46-KO cl1 or cl2 fibroblasts ( P = 0.9905, 0.9968, 0.2401, 0.8080, 0.0067, and 0.0065, respectively). ** P < 0.01; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( E , F ) WB ( E ) and densitometric quantification ( F ) of Itgb1 and Itga5 protein levels in WT and USP12/46-dKO fibroblasts. Gapdh served as loading control. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test comparing the WT fibroblasts with USP12/46-KO cl1 or cl2 fibroblasts (for Itgb1, P = 0.0162 and 0.0189, respectively; for Itga5, P = 0.0180 and 0.0106, respectively). * P < 0.05. Data were shown as Mean ± SD, n = 3 independent experiments. ( G – I ) Itgb1 surface levels were determined by flow cytometry ( G ), Itgb1 protein levels in cell lysates were determined by WB ( H ), and densitometric quantification ( I ) in WT and USP12/46-dKO fibroblasts stably expressing EGFP, EGFP-USP12 WT , or EGFP-USP12 C48S . Gapdh served as a loading control. Statistical analysis was carried out by RM two-way ANOVA with Dunnett’s multiple comparison test. In ( G ), statistical significance was tested comparing the EGFP group with EGFP-USP12 WT or EGFP-USP12 C48S group in WT fibroblasts ( P = 0.3449 and 0.0193, respectively); in USP12/46-dKO cl1 fibroblasts ( P = 0.0335 and 0.6230, respectively); and in USP12/46-dKO cl2 fibroblasts ( P = 0.0184 and 0.9260, respectively). In ( I ), statistical significance was tested comparing the EGFP group with EGFP-USP12 WT or EGFP-USP12 C48S group in WT fibroblasts ( P = 0.6467 and 0.6716, respectively); in USP12/46-dKO cl1 fibroblasts ( P = 0.0266 and 0.3351, respectively); and in USP12/46-dKO cl2 fibroblasts ( P = 0.0332 and 0.1310, respectively). * P < 0.05; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( J ) Volcano plot of the cell surface proteome of USP12/46-dKO fibroblasts stably expressing EGFP-USP12 C48S versus EGFP-USP12 WT identified by label-free MS. P values were determined using two-sided permuted t -test with 250 randomizations. The black dashed line indicates the significance cutoff (FDR:0.05, S0:0.1) estimated by the Perseus software. n = 4 biological replicates. Arbitrarily selected cell surface receptors were highlighted in red. .
Pacyc184 Pa15a Origin Replication, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pacyc184 pa15a origin replication/product/Millipore
Average 90 stars, based on 1 article reviews
pacyc184 pa15a origin replication - by Bioz Stars, 2026-03
90/100 stars
  Buy from Supplier
pmr1 6 × his-sec7δc (residues 203–1017  (Millipore)
90
Millipore pmr1 6 × his-sec7δc (residues 203–1017
The HUS-box mutation does not prevent N-terminal dimerization. A, schematic of Sec7 constructs used in this study. The red star indicates the location of the N653A HUS-box mutation. Sec7f, residues 203–2009; <t>Sec7ΔC+HDS1,</t> residues 203–1220; Sec7ΔC, residues 203–1017. B, left panel, recombinant Sec7f WT and Sec7f N653A were purified from insect cells, resolved on an 8% SDS-PAGE gel, and then Coomassie-stained. Right panel, recombinant Sec7ΔC+HDS1 (WT and N653A), Sec7ΔC (WT and N653A), myristoylated Arf1, and ΔN17-Arf1 were purified from E. coli and resolved on a 15% SDS-PAGE gel. The asterisk indicates a contaminant. C, MALS coupled to gel filtration results for Sec7ΔC WT (left panel) and N653A mutant (right panel). Both constructs are dimeric.
Pmr1 6 × His Sec7δc (Residues 203–1017, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pmr1 6 × his-sec7δc (residues 203–1017/product/Millipore
Average 90 stars, based on 1 article reviews
pmr1 6 × his-sec7δc (residues 203–1017 - by Bioz Stars, 2026-03
90/100 stars
  Buy from Supplier
by4741  (Thermo Fisher)
90
Thermo Fisher by4741
The HUS-box mutation does not prevent N-terminal dimerization. A, schematic of Sec7 constructs used in this study. The red star indicates the location of the N653A HUS-box mutation. Sec7f, residues 203–2009; <t>Sec7ΔC+HDS1,</t> residues 203–1220; Sec7ΔC, residues 203–1017. B, left panel, recombinant Sec7f WT and Sec7f N653A were purified from insect cells, resolved on an 8% SDS-PAGE gel, and then Coomassie-stained. Right panel, recombinant Sec7ΔC+HDS1 (WT and N653A), Sec7ΔC (WT and N653A), myristoylated Arf1, and ΔN17-Arf1 were purified from E. coli and resolved on a 15% SDS-PAGE gel. The asterisk indicates a contaminant. C, MALS coupled to gel filtration results for Sec7ΔC WT (left panel) and N653A mutant (right panel). Both constructs are dimeric.
By4741, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/by4741/product/Thermo Fisher
Average 90 stars, based on 1 article reviews
by4741 - by Bioz Stars, 2026-03
90/100 stars
  Buy from Supplier

Image Search Results


Yeast strains used in this study

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antagonistic roles for the ubiquitin ligase Asr1 and the ubiquitin-specific protease Ubp3 in subtelomeric gene silencing

doi: 10.1073/pnas.1518375113

Figure Lengend Snippet: Yeast strains used in this study

Article Snippet: Yeast strains used in this study table ft1 table-wrap mode="anchored" t5 Table S3. caption a7 Plasmid Description Source pYES2 GAL1pr / URA3 /2µ vector ThermoFisher pYES2 HA-Asr1 pYES2 but HA- ASR1 ( 5 ) pYES2 HA-Asr1 RING pYES2 but HA- asr1 C26A/C29A/C66A/C69A ( 5 ) pYES2 HA-Asr1 PHD pYES2 but HA- asr1 C143A/C146A/C186A/C189A ( 5 ) pYES2 HA-CBD pYES2 but HA- asr1 N198∆ ( 5 ) pRS415 GPD GPD1pr / LEU2 /CEN vector ( 26 ) pRS415 Ubp3-3MYC pRS415 GPD but UBP3 -3Myc This study pRS415 Ubp3-3MYC N45∆ pRS415 GPD but upb3 -3Myc N45∆ This study pRS415 Ubp3-3MYC N90∆ pRS415 GPD but upb3 -3Myc N90∆ This study pRS415 Ubp3-3MYC N145∆ pRS415 GPD but upb3 -3Myc N145∆ This study pRS415 Ubp3-3MYC N180∆ pRS415 GPD but upb3 -3Myc N180∆ This study pRS415 HA-Asr1 pRS415 GPD but HA-ASR1 This study pRS415 HA-Asr1 RING pRS415 GPD but HA- asr1 C26A/C29A/C66A/C69A This study pRS415 HA-Asr1 PHD pRS415 GPD but HA- asr1 C143A/C146A/C186A/C189A This study pRS316 URA3/ CEN ATCC 77145 pUB221 CUP1pr::6xHIS-MYC-Ubi/TRP1 and URA3 /2µ vector ( 32 ) pRS415 N180Ubp3-GFP-3MYC pRS415 GPD but GFP-N180 UBP3 -3Myc This study pRS415 GFP-3MYC pRS415 GPD but GFP-3Myc This study Open in a separate window Plasmids used in this study

Techniques:

Validated proteins associated with Asr1-TAP

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antagonistic roles for the ubiquitin ligase Asr1 and the ubiquitin-specific protease Ubp3 in subtelomeric gene silencing

doi: 10.1073/pnas.1518375113

Figure Lengend Snippet: Validated proteins associated with Asr1-TAP

Article Snippet: Yeast strains used in this study table ft1 table-wrap mode="anchored" t5 Table S3. caption a7 Plasmid Description Source pYES2 GAL1pr / URA3 /2µ vector ThermoFisher pYES2 HA-Asr1 pYES2 but HA- ASR1 ( 5 ) pYES2 HA-Asr1 RING pYES2 but HA- asr1 C26A/C29A/C66A/C69A ( 5 ) pYES2 HA-Asr1 PHD pYES2 but HA- asr1 C143A/C146A/C186A/C189A ( 5 ) pYES2 HA-CBD pYES2 but HA- asr1 N198∆ ( 5 ) pRS415 GPD GPD1pr / LEU2 /CEN vector ( 26 ) pRS415 Ubp3-3MYC pRS415 GPD but UBP3 -3Myc This study pRS415 Ubp3-3MYC N45∆ pRS415 GPD but upb3 -3Myc N45∆ This study pRS415 Ubp3-3MYC N90∆ pRS415 GPD but upb3 -3Myc N90∆ This study pRS415 Ubp3-3MYC N145∆ pRS415 GPD but upb3 -3Myc N145∆ This study pRS415 Ubp3-3MYC N180∆ pRS415 GPD but upb3 -3Myc N180∆ This study pRS415 HA-Asr1 pRS415 GPD but HA-ASR1 This study pRS415 HA-Asr1 RING pRS415 GPD but HA- asr1 C26A/C29A/C66A/C69A This study pRS415 HA-Asr1 PHD pRS415 GPD but HA- asr1 C143A/C146A/C186A/C189A This study pRS316 URA3/ CEN ATCC 77145 pUB221 CUP1pr::6xHIS-MYC-Ubi/TRP1 and URA3 /2µ vector ( 32 ) pRS415 N180Ubp3-GFP-3MYC pRS415 GPD but GFP-N180 UBP3 -3Myc This study pRS415 GFP-3MYC pRS415 GPD but GFP-3Myc This study Open in a separate window Plasmids used in this study

Techniques:

Asr1 associates with the Ubp3/Bre5 deubiquitylase. (A) Asr1 coprecipitates with Ubp3. Extract was prepared from cells expressing HA-tagged Ubp3, either alone (YTM1) or in conjunction with FLAG (FL)-tagged Asr1 (YTM2). Immunoprecipitation (IP) was performed with an anti-FLAG antibody (F-IP), and products were subjected to immunoblotting (IB) with anti-HA and -FLAG antibodies. For Ubp3–HA, 1% of the input (inp) to the IP was also analyzed by IB; for Asr1–FLAG, 7.5% of the input was analyzed. (B) Asr1 coprecipitates with Bre5. Extract was prepared from cells expressing FLAG (FL)-tagged Bre5, either alone (YTM3) or in conjunction with HA-tagged Asr1 (YTM4). IP was performed with an anti-HA antibody (H-IP), and products were subjected to IB with anti-HA and -FLAG antibodies. For Bre5–FL, 1% of the input (inp) to the IP was analyzed by IB; for Asr1–HA, 7.5% of the input was analyzed. (C) The amino terminus of Asr1 is required for association with Ubp3. Extract was prepared from yeast cells expressing galactose-inducible HA-tagged Asr1 proteins, either alone (WT, YTM9; Asr1RINGm, YTM10; Asr1PHDm, YTM11; CBD, YTM12) or in the presence of a plasmid expressing MYC-tagged Ubp3 (WT, YTM13; Asr1RINGm, YTM14; Asr1PHDm,YTM15; CBD, YTM16). IP was performed with an anti-MYC antibody (MYC-IP), and products were subjected to IB with anti-HA and -MYC antibodies. The * indicates a low molecular weight MYC-reactive species that we assume is a degradation product of Ubp3 that forms during the IP. (D) The amino terminus of Ubp3 mediates interaction with Asr1. Extract was prepared from yeast cells expressing galactose-inducible HA-tagged Asr1 and FLAG-tagged Bre5, either alone (–, YTM9) or in the presence of a plasmid expressing MYC–tagged Ubp3 proteins (WT, YTM13; ∆N180, YTM17; ∆N145, YTM18; ∆N90, YTM19; ∆N45, YTM20). IP was performed with an anti-MYC antibody (MYC-IP), and products were subjected to IB with anti-HA, -FLAG, and -MYC antibodies.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antagonistic roles for the ubiquitin ligase Asr1 and the ubiquitin-specific protease Ubp3 in subtelomeric gene silencing

doi: 10.1073/pnas.1518375113

Figure Lengend Snippet: Asr1 associates with the Ubp3/Bre5 deubiquitylase. (A) Asr1 coprecipitates with Ubp3. Extract was prepared from cells expressing HA-tagged Ubp3, either alone (YTM1) or in conjunction with FLAG (FL)-tagged Asr1 (YTM2). Immunoprecipitation (IP) was performed with an anti-FLAG antibody (F-IP), and products were subjected to immunoblotting (IB) with anti-HA and -FLAG antibodies. For Ubp3–HA, 1% of the input (inp) to the IP was also analyzed by IB; for Asr1–FLAG, 7.5% of the input was analyzed. (B) Asr1 coprecipitates with Bre5. Extract was prepared from cells expressing FLAG (FL)-tagged Bre5, either alone (YTM3) or in conjunction with HA-tagged Asr1 (YTM4). IP was performed with an anti-HA antibody (H-IP), and products were subjected to IB with anti-HA and -FLAG antibodies. For Bre5–FL, 1% of the input (inp) to the IP was analyzed by IB; for Asr1–HA, 7.5% of the input was analyzed. (C) The amino terminus of Asr1 is required for association with Ubp3. Extract was prepared from yeast cells expressing galactose-inducible HA-tagged Asr1 proteins, either alone (WT, YTM9; Asr1RINGm, YTM10; Asr1PHDm, YTM11; CBD, YTM12) or in the presence of a plasmid expressing MYC-tagged Ubp3 (WT, YTM13; Asr1RINGm, YTM14; Asr1PHDm,YTM15; CBD, YTM16). IP was performed with an anti-MYC antibody (MYC-IP), and products were subjected to IB with anti-HA and -MYC antibodies. The * indicates a low molecular weight MYC-reactive species that we assume is a degradation product of Ubp3 that forms during the IP. (D) The amino terminus of Ubp3 mediates interaction with Asr1. Extract was prepared from yeast cells expressing galactose-inducible HA-tagged Asr1 and FLAG-tagged Bre5, either alone (–, YTM9) or in the presence of a plasmid expressing MYC–tagged Ubp3 proteins (WT, YTM13; ∆N180, YTM17; ∆N145, YTM18; ∆N90, YTM19; ∆N45, YTM20). IP was performed with an anti-MYC antibody (MYC-IP), and products were subjected to IB with anti-HA, -FLAG, and -MYC antibodies.

Article Snippet: Yeast strains used in this study table ft1 table-wrap mode="anchored" t5 Table S3. caption a7 Plasmid Description Source pYES2 GAL1pr / URA3 /2µ vector ThermoFisher pYES2 HA-Asr1 pYES2 but HA- ASR1 ( 5 ) pYES2 HA-Asr1 RING pYES2 but HA- asr1 C26A/C29A/C66A/C69A ( 5 ) pYES2 HA-Asr1 PHD pYES2 but HA- asr1 C143A/C146A/C186A/C189A ( 5 ) pYES2 HA-CBD pYES2 but HA- asr1 N198∆ ( 5 ) pRS415 GPD GPD1pr / LEU2 /CEN vector ( 26 ) pRS415 Ubp3-3MYC pRS415 GPD but UBP3 -3Myc This study pRS415 Ubp3-3MYC N45∆ pRS415 GPD but upb3 -3Myc N45∆ This study pRS415 Ubp3-3MYC N90∆ pRS415 GPD but upb3 -3Myc N90∆ This study pRS415 Ubp3-3MYC N145∆ pRS415 GPD but upb3 -3Myc N145∆ This study pRS415 Ubp3-3MYC N180∆ pRS415 GPD but upb3 -3Myc N180∆ This study pRS415 HA-Asr1 pRS415 GPD but HA-ASR1 This study pRS415 HA-Asr1 RING pRS415 GPD but HA- asr1 C26A/C29A/C66A/C69A This study pRS415 HA-Asr1 PHD pRS415 GPD but HA- asr1 C143A/C146A/C186A/C189A This study pRS316 URA3/ CEN ATCC 77145 pUB221 CUP1pr::6xHIS-MYC-Ubi/TRP1 and URA3 /2µ vector ( 32 ) pRS415 N180Ubp3-GFP-3MYC pRS415 GPD but GFP-N180 UBP3 -3Myc This study pRS415 GFP-3MYC pRS415 GPD but GFP-3Myc This study Open in a separate window Plasmids used in this study

Techniques: Expressing, Immunoprecipitation, Western Blot, Plasmid Preparation, Molecular Weight

Schematic of Ubp3. Shown are the location of the domains required for interaction with Bre5 and for catalysis. Beneath the schematic is a scaled representation of the location of amino-terminal truncation mutants characterized for interaction with Asr1 in Fig. 2D.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antagonistic roles for the ubiquitin ligase Asr1 and the ubiquitin-specific protease Ubp3 in subtelomeric gene silencing

doi: 10.1073/pnas.1518375113

Figure Lengend Snippet: Schematic of Ubp3. Shown are the location of the domains required for interaction with Bre5 and for catalysis. Beneath the schematic is a scaled representation of the location of amino-terminal truncation mutants characterized for interaction with Asr1 in Fig. 2D.

Article Snippet: Yeast strains used in this study table ft1 table-wrap mode="anchored" t5 Table S3. caption a7 Plasmid Description Source pYES2 GAL1pr / URA3 /2µ vector ThermoFisher pYES2 HA-Asr1 pYES2 but HA- ASR1 ( 5 ) pYES2 HA-Asr1 RING pYES2 but HA- asr1 C26A/C29A/C66A/C69A ( 5 ) pYES2 HA-Asr1 PHD pYES2 but HA- asr1 C143A/C146A/C186A/C189A ( 5 ) pYES2 HA-CBD pYES2 but HA- asr1 N198∆ ( 5 ) pRS415 GPD GPD1pr / LEU2 /CEN vector ( 26 ) pRS415 Ubp3-3MYC pRS415 GPD but UBP3 -3Myc This study pRS415 Ubp3-3MYC N45∆ pRS415 GPD but upb3 -3Myc N45∆ This study pRS415 Ubp3-3MYC N90∆ pRS415 GPD but upb3 -3Myc N90∆ This study pRS415 Ubp3-3MYC N145∆ pRS415 GPD but upb3 -3Myc N145∆ This study pRS415 Ubp3-3MYC N180∆ pRS415 GPD but upb3 -3Myc N180∆ This study pRS415 HA-Asr1 pRS415 GPD but HA-ASR1 This study pRS415 HA-Asr1 RING pRS415 GPD but HA- asr1 C26A/C29A/C66A/C69A This study pRS415 HA-Asr1 PHD pRS415 GPD but HA- asr1 C143A/C146A/C186A/C189A This study pRS316 URA3/ CEN ATCC 77145 pUB221 CUP1pr::6xHIS-MYC-Ubi/TRP1 and URA3 /2µ vector ( 32 ) pRS415 N180Ubp3-GFP-3MYC pRS415 GPD but GFP-N180 UBP3 -3Myc This study pRS415 GFP-3MYC pRS415 GPD but GFP-3Myc This study Open in a separate window Plasmids used in this study

Techniques:

Interaction of Ubp3 with RNA polymerase II is mediated via Asr1. (A) The amino terminus of Ubp3 suffices for interaction with Asr1 and pol II. Extract was prepared from cells expressing HA-tagged Asr1, FLAG-tagged Rpb3, and either MYC-tagged GFP alone (YTM43), or MYC-tagged GFP fused to the amino-terminal 180 residues of Ubp3 (YTM42). Immunoprecipitation (IP) was performed with an anti-MYC antibody (M-IP), and products were subjected to immunoblotting (IB) with anti-HA, -FLAG, and -MYC antibodies. For Rpb3–FLAG and Asr1–HA, 2.5% of the input was analyzed; for Ubp3–MYC, 0.1% of the input was analyzed. (B) Ubp3 requires Asr1 to interact with pol II. Extracts were prepared from yeast expressing FLAG-tagged Rpb3, and carrying combinations of (i) an ASR1 gene deletion (∆) or expression of WT HA-tagged Asr1, and (ii) WT MYC-tagged Ubp3 (WT) or the ∆N180 MYC-tagged Ubp3 mutant (lanes 1 and 7, YTM21; lanes 2 and 8, YTM22; lanes 3 and 9, YTM23; lanes 4 and 10, YTM24; lanes 5 and 11, YTM25; lanes 6 and 12, YTM26). IP was performed with an anti-MYC antibody (MYC-IP), and products were subjected to IB with anti-pSer5, -FLAG, -HA, and -MYC antibodies. For pSer5, Rpb3–FLAG, and Asr1–HA, 2.5% of the input was analyzed; for Ubp3–MYC, 0.1% of the input was analyzed.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antagonistic roles for the ubiquitin ligase Asr1 and the ubiquitin-specific protease Ubp3 in subtelomeric gene silencing

doi: 10.1073/pnas.1518375113

Figure Lengend Snippet: Interaction of Ubp3 with RNA polymerase II is mediated via Asr1. (A) The amino terminus of Ubp3 suffices for interaction with Asr1 and pol II. Extract was prepared from cells expressing HA-tagged Asr1, FLAG-tagged Rpb3, and either MYC-tagged GFP alone (YTM43), or MYC-tagged GFP fused to the amino-terminal 180 residues of Ubp3 (YTM42). Immunoprecipitation (IP) was performed with an anti-MYC antibody (M-IP), and products were subjected to immunoblotting (IB) with anti-HA, -FLAG, and -MYC antibodies. For Rpb3–FLAG and Asr1–HA, 2.5% of the input was analyzed; for Ubp3–MYC, 0.1% of the input was analyzed. (B) Ubp3 requires Asr1 to interact with pol II. Extracts were prepared from yeast expressing FLAG-tagged Rpb3, and carrying combinations of (i) an ASR1 gene deletion (∆) or expression of WT HA-tagged Asr1, and (ii) WT MYC-tagged Ubp3 (WT) or the ∆N180 MYC-tagged Ubp3 mutant (lanes 1 and 7, YTM21; lanes 2 and 8, YTM22; lanes 3 and 9, YTM23; lanes 4 and 10, YTM24; lanes 5 and 11, YTM25; lanes 6 and 12, YTM26). IP was performed with an anti-MYC antibody (MYC-IP), and products were subjected to IB with anti-pSer5, -FLAG, -HA, and -MYC antibodies. For pSer5, Rpb3–FLAG, and Asr1–HA, 2.5% of the input was analyzed; for Ubp3–MYC, 0.1% of the input was analyzed.

Article Snippet: Yeast strains used in this study table ft1 table-wrap mode="anchored" t5 Table S3. caption a7 Plasmid Description Source pYES2 GAL1pr / URA3 /2µ vector ThermoFisher pYES2 HA-Asr1 pYES2 but HA- ASR1 ( 5 ) pYES2 HA-Asr1 RING pYES2 but HA- asr1 C26A/C29A/C66A/C69A ( 5 ) pYES2 HA-Asr1 PHD pYES2 but HA- asr1 C143A/C146A/C186A/C189A ( 5 ) pYES2 HA-CBD pYES2 but HA- asr1 N198∆ ( 5 ) pRS415 GPD GPD1pr / LEU2 /CEN vector ( 26 ) pRS415 Ubp3-3MYC pRS415 GPD but UBP3 -3Myc This study pRS415 Ubp3-3MYC N45∆ pRS415 GPD but upb3 -3Myc N45∆ This study pRS415 Ubp3-3MYC N90∆ pRS415 GPD but upb3 -3Myc N90∆ This study pRS415 Ubp3-3MYC N145∆ pRS415 GPD but upb3 -3Myc N145∆ This study pRS415 Ubp3-3MYC N180∆ pRS415 GPD but upb3 -3Myc N180∆ This study pRS415 HA-Asr1 pRS415 GPD but HA-ASR1 This study pRS415 HA-Asr1 RING pRS415 GPD but HA- asr1 C26A/C29A/C66A/C69A This study pRS415 HA-Asr1 PHD pRS415 GPD but HA- asr1 C143A/C146A/C186A/C189A This study pRS316 URA3/ CEN ATCC 77145 pUB221 CUP1pr::6xHIS-MYC-Ubi/TRP1 and URA3 /2µ vector ( 32 ) pRS415 N180Ubp3-GFP-3MYC pRS415 GPD but GFP-N180 UBP3 -3Myc This study pRS415 GFP-3MYC pRS415 GPD but GFP-3Myc This study Open in a separate window Plasmids used in this study

Techniques: Expressing, Immunoprecipitation, Western Blot, Mutagenesis

Lack of genetic interaction between ASR1 and UBP3 in modulating sensitivity to chemical agents. (A–C) Analysis of interactions between ∆ubp3 and the ASR1 null or RING finger mutations. Fivefold serial dilutions of the indicated yeast strains (ASR1:UBP3, BY4741; ∆asr1:UBP3, YTM5; ASR1-RINGm:UBP3, YTM27; ASR1:∆ubp3, ∆Ubp3; ∆asr1:∆ubp3, YTM6; ASR1-RINGm:∆ubp3, YTM28) were spotted on plates containing (A) 150 μg/mL 6-AU, (B) 0.03% MMS, or (C) 5 nM Rapamycin, or relevant vehicle control, and grown for 2–3 d before being photographed. (D–F) Analysis of the impact of mutations in Ubp3 that disrupt interaction with Asr1. Fivefold serial dilutions of the indicated yeast strains (UBP3:vector, YTM44; ∆ubp3:vector, YTM45; ∆ubp3:UBP3-WT, YTM46; ∆ubp3:UBP3-∆N180, YTM47; ∆ubp3:UBP3-∆N145, YTM48) were spotted on plates containing (D) 100 μg/mL 6-AU, (E) 0.02% MMS, or (F) 1 nM Rapamycin, or relevant vehicle control, and grown for 2–3 d before being photographed. See Table S2 for strain details.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antagonistic roles for the ubiquitin ligase Asr1 and the ubiquitin-specific protease Ubp3 in subtelomeric gene silencing

doi: 10.1073/pnas.1518375113

Figure Lengend Snippet: Lack of genetic interaction between ASR1 and UBP3 in modulating sensitivity to chemical agents. (A–C) Analysis of interactions between ∆ubp3 and the ASR1 null or RING finger mutations. Fivefold serial dilutions of the indicated yeast strains (ASR1:UBP3, BY4741; ∆asr1:UBP3, YTM5; ASR1-RINGm:UBP3, YTM27; ASR1:∆ubp3, ∆Ubp3; ∆asr1:∆ubp3, YTM6; ASR1-RINGm:∆ubp3, YTM28) were spotted on plates containing (A) 150 μg/mL 6-AU, (B) 0.03% MMS, or (C) 5 nM Rapamycin, or relevant vehicle control, and grown for 2–3 d before being photographed. (D–F) Analysis of the impact of mutations in Ubp3 that disrupt interaction with Asr1. Fivefold serial dilutions of the indicated yeast strains (UBP3:vector, YTM44; ∆ubp3:vector, YTM45; ∆ubp3:UBP3-WT, YTM46; ∆ubp3:UBP3-∆N180, YTM47; ∆ubp3:UBP3-∆N145, YTM48) were spotted on plates containing (D) 100 μg/mL 6-AU, (E) 0.02% MMS, or (F) 1 nM Rapamycin, or relevant vehicle control, and grown for 2–3 d before being photographed. See Table S2 for strain details.

Article Snippet: Yeast strains used in this study table ft1 table-wrap mode="anchored" t5 Table S3. caption a7 Plasmid Description Source pYES2 GAL1pr / URA3 /2µ vector ThermoFisher pYES2 HA-Asr1 pYES2 but HA- ASR1 ( 5 ) pYES2 HA-Asr1 RING pYES2 but HA- asr1 C26A/C29A/C66A/C69A ( 5 ) pYES2 HA-Asr1 PHD pYES2 but HA- asr1 C143A/C146A/C186A/C189A ( 5 ) pYES2 HA-CBD pYES2 but HA- asr1 N198∆ ( 5 ) pRS415 GPD GPD1pr / LEU2 /CEN vector ( 26 ) pRS415 Ubp3-3MYC pRS415 GPD but UBP3 -3Myc This study pRS415 Ubp3-3MYC N45∆ pRS415 GPD but upb3 -3Myc N45∆ This study pRS415 Ubp3-3MYC N90∆ pRS415 GPD but upb3 -3Myc N90∆ This study pRS415 Ubp3-3MYC N145∆ pRS415 GPD but upb3 -3Myc N145∆ This study pRS415 Ubp3-3MYC N180∆ pRS415 GPD but upb3 -3Myc N180∆ This study pRS415 HA-Asr1 pRS415 GPD but HA-ASR1 This study pRS415 HA-Asr1 RING pRS415 GPD but HA- asr1 C26A/C29A/C66A/C69A This study pRS415 HA-Asr1 PHD pRS415 GPD but HA- asr1 C143A/C146A/C186A/C189A This study pRS316 URA3/ CEN ATCC 77145 pUB221 CUP1pr::6xHIS-MYC-Ubi/TRP1 and URA3 /2µ vector ( 32 ) pRS415 N180Ubp3-GFP-3MYC pRS415 GPD but GFP-N180 UBP3 -3Myc This study pRS415 GFP-3MYC pRS415 GPD but GFP-3Myc This study Open in a separate window Plasmids used in this study

Techniques: Plasmid Preparation

Asr1 and Ubp3 act antagonistically in silencing of subtelomeric chromatin. (A) Deletion of ASR1 attenuates the impact of UBP3 deletion on silencing. Equal amounts of yeast (WT, LPY4819; ∆asr1, LPY4819 ∆Asr1; ∆ubp3, LPY4819 ∆Ubp3; ∆ubp3∆asr1, LPY4819 ∆Asr1 ∆Ubp3; ∆sir2, LPY4977) were plated on media with or without 5-FOA, and colonies were counted. Error bars represent SEM (n = 3). (B) Loss of ASR1 is epistatic to loss of UBP3. Yeast (WT, YPH499UTAT; ∆asr1, UTAT TM1; ∆ubp3, UTAT TM2; ∆asr1∆ubp3, UTAT TM3; ∆sir2, UTAT TM12) were plated on nonselective media (CSM), and the ratio of white and red colonies for each plate was calculated. Error bars represent SEM (n = 3). (C) Deletion of UBP3 reverses the impact of the Asr1RING mutation deletion on silencing. Total RNA was extracted from yeast (WT, BY4741; ∆asr1, YTM5; asr1RINGm, YTM27; ∆ubp3, ∆Ubp3; ∆asr1∆ubp3, YTM6; asr1RINGm∆ubp3, YTM28), and RT–QPCR was used to measure transcript levels from the indicated loci. The expression level of each gene in WT (WT) congenic cells was set to one. Error bars represent SEM (n = 8). (D) Asr1 and Ubp3 oppositely impact ubiquitylation of serine 5-phosphorylated Rpb1. Denaturing extracts were prepared from a WT yeast strain (lane 1, YTM31), or yeast expressing polyhistidine-tagged ubiquitin (His–Ub) and carrying the indicated genotype (WT, YTM32; ∆ubp3, YTM35; ∆asr1, YTM33; ∆asr1∆ubp3, YTM36; asr1RINGm, YTM34; asr1RINGm∆ubp3, YTM37). Ubiquitylated proteins were recovered on Ni-NTA resin. Levels of serine 5-phosphorylated Rpb1 in the Ni-NTA–bound material and a sample of the input were detected by immunoblotting with a pSer5-specific antibody (pSer5 IB). (E) Steady-state levels of Ubp3 deletion mutants assayed in F. Ubp3 was MYC-tagged and visualized by an anti-MYC IB. An antibody against actin was used as a loading control. (F). The amino terminus of Ubp3 is required for its prosilencing function. Yeast cells (WT, UTAT TM4; ∆ubp3, UTAT TM9; ∆ubp3UBP3, UTAT TM10; ∆ubp3∆N180, UTAT TM11) were plated to single colony density on leucine drop-out media, and the ratio of white and red colonies for each plate was calculated. Error bars represent SEM (n = 3). (G) The amino terminus of Ubp3 is required for its effects on ubiquitylation of serine 5-phosphorylated Rpb1. A His–Ub assay, as in D, was performed on a WT yeast strain (lane 1, YTM49) or yeast expressing polyhistidine-tagged ubiquitin (His–Ub) and carrying the indicated genotype (WT, YTM50; ∆ubp3, YTM51; +UBP3, YTM52; +∆N180, YTM53; ∆ubp3/RINGm, YTM54; RINGm/+UBP3, YTM55; RINGm/+N180∆, YTM56).

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antagonistic roles for the ubiquitin ligase Asr1 and the ubiquitin-specific protease Ubp3 in subtelomeric gene silencing

doi: 10.1073/pnas.1518375113

Figure Lengend Snippet: Asr1 and Ubp3 act antagonistically in silencing of subtelomeric chromatin. (A) Deletion of ASR1 attenuates the impact of UBP3 deletion on silencing. Equal amounts of yeast (WT, LPY4819; ∆asr1, LPY4819 ∆Asr1; ∆ubp3, LPY4819 ∆Ubp3; ∆ubp3∆asr1, LPY4819 ∆Asr1 ∆Ubp3; ∆sir2, LPY4977) were plated on media with or without 5-FOA, and colonies were counted. Error bars represent SEM (n = 3). (B) Loss of ASR1 is epistatic to loss of UBP3. Yeast (WT, YPH499UTAT; ∆asr1, UTAT TM1; ∆ubp3, UTAT TM2; ∆asr1∆ubp3, UTAT TM3; ∆sir2, UTAT TM12) were plated on nonselective media (CSM), and the ratio of white and red colonies for each plate was calculated. Error bars represent SEM (n = 3). (C) Deletion of UBP3 reverses the impact of the Asr1RING mutation deletion on silencing. Total RNA was extracted from yeast (WT, BY4741; ∆asr1, YTM5; asr1RINGm, YTM27; ∆ubp3, ∆Ubp3; ∆asr1∆ubp3, YTM6; asr1RINGm∆ubp3, YTM28), and RT–QPCR was used to measure transcript levels from the indicated loci. The expression level of each gene in WT (WT) congenic cells was set to one. Error bars represent SEM (n = 8). (D) Asr1 and Ubp3 oppositely impact ubiquitylation of serine 5-phosphorylated Rpb1. Denaturing extracts were prepared from a WT yeast strain (lane 1, YTM31), or yeast expressing polyhistidine-tagged ubiquitin (His–Ub) and carrying the indicated genotype (WT, YTM32; ∆ubp3, YTM35; ∆asr1, YTM33; ∆asr1∆ubp3, YTM36; asr1RINGm, YTM34; asr1RINGm∆ubp3, YTM37). Ubiquitylated proteins were recovered on Ni-NTA resin. Levels of serine 5-phosphorylated Rpb1 in the Ni-NTA–bound material and a sample of the input were detected by immunoblotting with a pSer5-specific antibody (pSer5 IB). (E) Steady-state levels of Ubp3 deletion mutants assayed in F. Ubp3 was MYC-tagged and visualized by an anti-MYC IB. An antibody against actin was used as a loading control. (F). The amino terminus of Ubp3 is required for its prosilencing function. Yeast cells (WT, UTAT TM4; ∆ubp3, UTAT TM9; ∆ubp3UBP3, UTAT TM10; ∆ubp3∆N180, UTAT TM11) were plated to single colony density on leucine drop-out media, and the ratio of white and red colonies for each plate was calculated. Error bars represent SEM (n = 3). (G) The amino terminus of Ubp3 is required for its effects on ubiquitylation of serine 5-phosphorylated Rpb1. A His–Ub assay, as in D, was performed on a WT yeast strain (lane 1, YTM49) or yeast expressing polyhistidine-tagged ubiquitin (His–Ub) and carrying the indicated genotype (WT, YTM50; ∆ubp3, YTM51; +UBP3, YTM52; +∆N180, YTM53; ∆ubp3/RINGm, YTM54; RINGm/+UBP3, YTM55; RINGm/+N180∆, YTM56).

Article Snippet: Yeast strains used in this study table ft1 table-wrap mode="anchored" t5 Table S3. caption a7 Plasmid Description Source pYES2 GAL1pr / URA3 /2µ vector ThermoFisher pYES2 HA-Asr1 pYES2 but HA- ASR1 ( 5 ) pYES2 HA-Asr1 RING pYES2 but HA- asr1 C26A/C29A/C66A/C69A ( 5 ) pYES2 HA-Asr1 PHD pYES2 but HA- asr1 C143A/C146A/C186A/C189A ( 5 ) pYES2 HA-CBD pYES2 but HA- asr1 N198∆ ( 5 ) pRS415 GPD GPD1pr / LEU2 /CEN vector ( 26 ) pRS415 Ubp3-3MYC pRS415 GPD but UBP3 -3Myc This study pRS415 Ubp3-3MYC N45∆ pRS415 GPD but upb3 -3Myc N45∆ This study pRS415 Ubp3-3MYC N90∆ pRS415 GPD but upb3 -3Myc N90∆ This study pRS415 Ubp3-3MYC N145∆ pRS415 GPD but upb3 -3Myc N145∆ This study pRS415 Ubp3-3MYC N180∆ pRS415 GPD but upb3 -3Myc N180∆ This study pRS415 HA-Asr1 pRS415 GPD but HA-ASR1 This study pRS415 HA-Asr1 RING pRS415 GPD but HA- asr1 C26A/C29A/C66A/C69A This study pRS415 HA-Asr1 PHD pRS415 GPD but HA- asr1 C143A/C146A/C186A/C189A This study pRS316 URA3/ CEN ATCC 77145 pUB221 CUP1pr::6xHIS-MYC-Ubi/TRP1 and URA3 /2µ vector ( 32 ) pRS415 N180Ubp3-GFP-3MYC pRS415 GPD but GFP-N180 UBP3 -3Myc This study pRS415 GFP-3MYC pRS415 GPD but GFP-3Myc This study Open in a separate window Plasmids used in this study

Techniques: Mutagenesis, Quantitative RT-PCR, Expressing, Western Blot

Asr1 and Ubp3 control expression of a euchromatic gene also regulated by Sir2. Total RNA was extracted from yeast cells of the indicated genotype (WT, BY4741; ∆asr1, YTM5; asr1RINGm, YTM27; ∆ubp3, ∆Ubp3; ∆asr1∆ubp3, YTM6; asr1RINGm∆ubp3, YTM28; ∆sir2; ∆Sir2), and RT–QPCR was used to measure transcript levels from the indicated loci. ACT1 was used as control locus. The expression level of each gene in WT (WT) congenic cells was set to one. Error bars represent SEM (n = 7). See Table S2 for strain details.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antagonistic roles for the ubiquitin ligase Asr1 and the ubiquitin-specific protease Ubp3 in subtelomeric gene silencing

doi: 10.1073/pnas.1518375113

Figure Lengend Snippet: Asr1 and Ubp3 control expression of a euchromatic gene also regulated by Sir2. Total RNA was extracted from yeast cells of the indicated genotype (WT, BY4741; ∆asr1, YTM5; asr1RINGm, YTM27; ∆ubp3, ∆Ubp3; ∆asr1∆ubp3, YTM6; asr1RINGm∆ubp3, YTM28; ∆sir2; ∆Sir2), and RT–QPCR was used to measure transcript levels from the indicated loci. ACT1 was used as control locus. The expression level of each gene in WT (WT) congenic cells was set to one. Error bars represent SEM (n = 7). See Table S2 for strain details.

Article Snippet: Yeast strains used in this study table ft1 table-wrap mode="anchored" t5 Table S3. caption a7 Plasmid Description Source pYES2 GAL1pr / URA3 /2µ vector ThermoFisher pYES2 HA-Asr1 pYES2 but HA- ASR1 ( 5 ) pYES2 HA-Asr1 RING pYES2 but HA- asr1 C26A/C29A/C66A/C69A ( 5 ) pYES2 HA-Asr1 PHD pYES2 but HA- asr1 C143A/C146A/C186A/C189A ( 5 ) pYES2 HA-CBD pYES2 but HA- asr1 N198∆ ( 5 ) pRS415 GPD GPD1pr / LEU2 /CEN vector ( 26 ) pRS415 Ubp3-3MYC pRS415 GPD but UBP3 -3Myc This study pRS415 Ubp3-3MYC N45∆ pRS415 GPD but upb3 -3Myc N45∆ This study pRS415 Ubp3-3MYC N90∆ pRS415 GPD but upb3 -3Myc N90∆ This study pRS415 Ubp3-3MYC N145∆ pRS415 GPD but upb3 -3Myc N145∆ This study pRS415 Ubp3-3MYC N180∆ pRS415 GPD but upb3 -3Myc N180∆ This study pRS415 HA-Asr1 pRS415 GPD but HA-ASR1 This study pRS415 HA-Asr1 RING pRS415 GPD but HA- asr1 C26A/C29A/C66A/C69A This study pRS415 HA-Asr1 PHD pRS415 GPD but HA- asr1 C143A/C146A/C186A/C189A This study pRS316 URA3/ CEN ATCC 77145 pUB221 CUP1pr::6xHIS-MYC-Ubi/TRP1 and URA3 /2µ vector ( 32 ) pRS415 N180Ubp3-GFP-3MYC pRS415 GPD but GFP-N180 UBP3 -3Myc This study pRS415 GFP-3MYC pRS415 GPD but GFP-3Myc This study Open in a separate window Plasmids used in this study

Techniques: Expressing, Quantitative RT-PCR

Plasmids used in this study

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Antagonistic roles for the ubiquitin ligase Asr1 and the ubiquitin-specific protease Ubp3 in subtelomeric gene silencing

doi: 10.1073/pnas.1518375113

Figure Lengend Snippet: Plasmids used in this study

Article Snippet: Yeast strains used in this study table ft1 table-wrap mode="anchored" t5 Table S3. caption a7 Plasmid Description Source pYES2 GAL1pr / URA3 /2µ vector ThermoFisher pYES2 HA-Asr1 pYES2 but HA- ASR1 ( 5 ) pYES2 HA-Asr1 RING pYES2 but HA- asr1 C26A/C29A/C66A/C69A ( 5 ) pYES2 HA-Asr1 PHD pYES2 but HA- asr1 C143A/C146A/C186A/C189A ( 5 ) pYES2 HA-CBD pYES2 but HA- asr1 N198∆ ( 5 ) pRS415 GPD GPD1pr / LEU2 /CEN vector ( 26 ) pRS415 Ubp3-3MYC pRS415 GPD but UBP3 -3Myc This study pRS415 Ubp3-3MYC N45∆ pRS415 GPD but upb3 -3Myc N45∆ This study pRS415 Ubp3-3MYC N90∆ pRS415 GPD but upb3 -3Myc N90∆ This study pRS415 Ubp3-3MYC N145∆ pRS415 GPD but upb3 -3Myc N145∆ This study pRS415 Ubp3-3MYC N180∆ pRS415 GPD but upb3 -3Myc N180∆ This study pRS415 HA-Asr1 pRS415 GPD but HA-ASR1 This study pRS415 HA-Asr1 RING pRS415 GPD but HA- asr1 C26A/C29A/C66A/C69A This study pRS415 HA-Asr1 PHD pRS415 GPD but HA- asr1 C143A/C146A/C186A/C189A This study pRS316 URA3/ CEN ATCC 77145 pUB221 CUP1pr::6xHIS-MYC-Ubi/TRP1 and URA3 /2µ vector ( 32 ) pRS415 N180Ubp3-GFP-3MYC pRS415 GPD but GFP-N180 UBP3 -3Myc This study pRS415 GFP-3MYC pRS415 GPD but GFP-3Myc This study Open in a separate window Plasmids used in this study

Techniques: Plasmid Preparation

( A ) Schematic overview of the CRISPR screen for identifying DUBs regulating Itgb1 surface levels. Cas9-expressing HAP1 cells were transduced with pooled lentiviral guide RNA (gRNA) libraries targeting 98 DUBs from the human genome. After 2 weeks in culture, cells with the 5% lowest (Itgb1 Lo ) and the 5% highest (Itgb1 Hi ) Itgb1 surface levels were sorted by flow cytometry, and gRNA-targeted genes were determined. ( B ) Volcano plot of the results from the CRISPR screen. The x-axis represents the log 2 fold change (lfc) in the frequency of genes targeted between the Itgb1 Lo and Itgb1 Hi populations. The y-axis indicates the robust rank aggregation (RRA) score determined by the MAGeCK algorithm (MAGeCK-RRA) (Li et al, ). Dots represent individual targeted genes, and those meeting the criteria of |lfc| >0.33 and −log 10 (RRA) >2 were considered significant. Genes significantly enriched in Itgb1 Lo cells are colored in blue, and those enriched in Itgb1 Hi in red. USP12 is marked in white. ( C ) Volcano plot of the α5β1 integrin proximitome determined by label-free MS analysis in mouse kidney fibroblasts expressing miniTurbo-tagged Itag5 (TurboID) versus Itgb1-KO fibroblasts (Ctrl). P values were determined using a two-sided permuted t -test with 250 randomizations. The black dashed line indicates the significance cutoff (FDR:0.05, S0:0.1) estimated by the Perseus software. n = 3 biological replicates. The red dots indicate the subunits of the α5β1 heterodimer and the components of the USP12/46-WDR48-WDR20 complex. ( D ) Itgb1 surface levels in WT and two independent clones (cl1 and cl2) of USP12-KO, USP46-KO, and USP12/46-dKO fibroblasts determined by flow cytometry. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test comparing the WT fibroblasts with USP12-KO cl1 or cl2, USP46-KO cl1 or cl2, USP12/46-KO cl1 or cl2 fibroblasts ( P = 0.9905, 0.9968, 0.2401, 0.8080, 0.0067, and 0.0065, respectively). ** P < 0.01; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( E , F ) WB ( E ) and densitometric quantification ( F ) of Itgb1 and Itga5 protein levels in WT and USP12/46-dKO fibroblasts. Gapdh served as loading control. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test comparing the WT fibroblasts with USP12/46-KO cl1 or cl2 fibroblasts (for Itgb1, P = 0.0162 and 0.0189, respectively; for Itga5, P = 0.0180 and 0.0106, respectively). * P < 0.05. Data were shown as Mean ± SD, n = 3 independent experiments. ( G – I ) Itgb1 surface levels were determined by flow cytometry ( G ), Itgb1 protein levels in cell lysates were determined by WB ( H ), and densitometric quantification ( I ) in WT and USP12/46-dKO fibroblasts stably expressing EGFP, EGFP-USP12 WT , or EGFP-USP12 C48S . Gapdh served as a loading control. Statistical analysis was carried out by RM two-way ANOVA with Dunnett’s multiple comparison test. In ( G ), statistical significance was tested comparing the EGFP group with EGFP-USP12 WT or EGFP-USP12 C48S group in WT fibroblasts ( P = 0.3449 and 0.0193, respectively); in USP12/46-dKO cl1 fibroblasts ( P = 0.0335 and 0.6230, respectively); and in USP12/46-dKO cl2 fibroblasts ( P = 0.0184 and 0.9260, respectively). In ( I ), statistical significance was tested comparing the EGFP group with EGFP-USP12 WT or EGFP-USP12 C48S group in WT fibroblasts ( P = 0.6467 and 0.6716, respectively); in USP12/46-dKO cl1 fibroblasts ( P = 0.0266 and 0.3351, respectively); and in USP12/46-dKO cl2 fibroblasts ( P = 0.0332 and 0.1310, respectively). * P < 0.05; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( J ) Volcano plot of the cell surface proteome of USP12/46-dKO fibroblasts stably expressing EGFP-USP12 C48S versus EGFP-USP12 WT identified by label-free MS. P values were determined using two-sided permuted t -test with 250 randomizations. The black dashed line indicates the significance cutoff (FDR:0.05, S0:0.1) estimated by the Perseus software. n = 4 biological replicates. Arbitrarily selected cell surface receptors were highlighted in red. .

Journal: EMBO Reports

Article Title: The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation

doi: 10.1038/s44319-024-00300-9

Figure Lengend Snippet: ( A ) Schematic overview of the CRISPR screen for identifying DUBs regulating Itgb1 surface levels. Cas9-expressing HAP1 cells were transduced with pooled lentiviral guide RNA (gRNA) libraries targeting 98 DUBs from the human genome. After 2 weeks in culture, cells with the 5% lowest (Itgb1 Lo ) and the 5% highest (Itgb1 Hi ) Itgb1 surface levels were sorted by flow cytometry, and gRNA-targeted genes were determined. ( B ) Volcano plot of the results from the CRISPR screen. The x-axis represents the log 2 fold change (lfc) in the frequency of genes targeted between the Itgb1 Lo and Itgb1 Hi populations. The y-axis indicates the robust rank aggregation (RRA) score determined by the MAGeCK algorithm (MAGeCK-RRA) (Li et al, ). Dots represent individual targeted genes, and those meeting the criteria of |lfc| >0.33 and −log 10 (RRA) >2 were considered significant. Genes significantly enriched in Itgb1 Lo cells are colored in blue, and those enriched in Itgb1 Hi in red. USP12 is marked in white. ( C ) Volcano plot of the α5β1 integrin proximitome determined by label-free MS analysis in mouse kidney fibroblasts expressing miniTurbo-tagged Itag5 (TurboID) versus Itgb1-KO fibroblasts (Ctrl). P values were determined using a two-sided permuted t -test with 250 randomizations. The black dashed line indicates the significance cutoff (FDR:0.05, S0:0.1) estimated by the Perseus software. n = 3 biological replicates. The red dots indicate the subunits of the α5β1 heterodimer and the components of the USP12/46-WDR48-WDR20 complex. ( D ) Itgb1 surface levels in WT and two independent clones (cl1 and cl2) of USP12-KO, USP46-KO, and USP12/46-dKO fibroblasts determined by flow cytometry. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test comparing the WT fibroblasts with USP12-KO cl1 or cl2, USP46-KO cl1 or cl2, USP12/46-KO cl1 or cl2 fibroblasts ( P = 0.9905, 0.9968, 0.2401, 0.8080, 0.0067, and 0.0065, respectively). ** P < 0.01; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( E , F ) WB ( E ) and densitometric quantification ( F ) of Itgb1 and Itga5 protein levels in WT and USP12/46-dKO fibroblasts. Gapdh served as loading control. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test comparing the WT fibroblasts with USP12/46-KO cl1 or cl2 fibroblasts (for Itgb1, P = 0.0162 and 0.0189, respectively; for Itga5, P = 0.0180 and 0.0106, respectively). * P < 0.05. Data were shown as Mean ± SD, n = 3 independent experiments. ( G – I ) Itgb1 surface levels were determined by flow cytometry ( G ), Itgb1 protein levels in cell lysates were determined by WB ( H ), and densitometric quantification ( I ) in WT and USP12/46-dKO fibroblasts stably expressing EGFP, EGFP-USP12 WT , or EGFP-USP12 C48S . Gapdh served as a loading control. Statistical analysis was carried out by RM two-way ANOVA with Dunnett’s multiple comparison test. In ( G ), statistical significance was tested comparing the EGFP group with EGFP-USP12 WT or EGFP-USP12 C48S group in WT fibroblasts ( P = 0.3449 and 0.0193, respectively); in USP12/46-dKO cl1 fibroblasts ( P = 0.0335 and 0.6230, respectively); and in USP12/46-dKO cl2 fibroblasts ( P = 0.0184 and 0.9260, respectively). In ( I ), statistical significance was tested comparing the EGFP group with EGFP-USP12 WT or EGFP-USP12 C48S group in WT fibroblasts ( P = 0.6467 and 0.6716, respectively); in USP12/46-dKO cl1 fibroblasts ( P = 0.0266 and 0.3351, respectively); and in USP12/46-dKO cl2 fibroblasts ( P = 0.0332 and 0.1310, respectively). * P < 0.05; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( J ) Volcano plot of the cell surface proteome of USP12/46-dKO fibroblasts stably expressing EGFP-USP12 C48S versus EGFP-USP12 WT identified by label-free MS. P values were determined using two-sided permuted t -test with 250 randomizations. The black dashed line indicates the significance cutoff (FDR:0.05, S0:0.1) estimated by the Perseus software. n = 4 biological replicates. Arbitrarily selected cell surface receptors were highlighted in red. .

Article Snippet: The cDNA encoding miniTurbo-tagged Itga5 was generated by fusing the miniTurbo tag (a gift from Alice Ting, Addgene plasmid # 107168) in frame to the 3′ end of the human Itag5 cDNA (a gift from Rick Horwitz, Addgene plasmid # 15238) and subsequently cloned into pRetroQ-N1 vector.

Techniques: CRISPR, Expressing, Transduction, Flow Cytometry, Software, Clone Assay, Comparison, Control, Stable Transfection

Reagents and tools table

Journal: EMBO Reports

Article Title: The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation

doi: 10.1038/s44319-024-00300-9

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The cDNA encoding miniTurbo-tagged Itga5 was generated by fusing the miniTurbo tag (a gift from Alice Ting, Addgene plasmid # 107168) in frame to the 3′ end of the human Itag5 cDNA (a gift from Rick Horwitz, Addgene plasmid # 15238) and subsequently cloned into pRetroQ-N1 vector.

Techniques: Recombinant, Expressing, Sequencing, Real-time Polymerase Chain Reaction, Protease Inhibitor, cDNA Synthesis, SYBR Green Assay, BIA-KA, Western Blot, Software, Microscopy

The HUS-box mutation does not prevent N-terminal dimerization. A, schematic of Sec7 constructs used in this study. The red star indicates the location of the N653A HUS-box mutation. Sec7f, residues 203–2009; Sec7ΔC+HDS1, residues 203–1220; Sec7ΔC, residues 203–1017. B, left panel, recombinant Sec7f WT and Sec7f N653A were purified from insect cells, resolved on an 8% SDS-PAGE gel, and then Coomassie-stained. Right panel, recombinant Sec7ΔC+HDS1 (WT and N653A), Sec7ΔC (WT and N653A), myristoylated Arf1, and ΔN17-Arf1 were purified from E. coli and resolved on a 15% SDS-PAGE gel. The asterisk indicates a contaminant. C, MALS coupled to gel filtration results for Sec7ΔC WT (left panel) and N653A mutant (right panel). Both constructs are dimeric.

Journal: The Journal of Biological Chemistry

Article Title: The HUS box is required for allosteric regulation of the Sec7 Arf-GEF

doi: 10.1074/jbc.RA117.001318

Figure Lengend Snippet: The HUS-box mutation does not prevent N-terminal dimerization. A, schematic of Sec7 constructs used in this study. The red star indicates the location of the N653A HUS-box mutation. Sec7f, residues 203–2009; Sec7ΔC+HDS1, residues 203–1220; Sec7ΔC, residues 203–1017. B, left panel, recombinant Sec7f WT and Sec7f N653A were purified from insect cells, resolved on an 8% SDS-PAGE gel, and then Coomassie-stained. Right panel, recombinant Sec7ΔC+HDS1 (WT and N653A), Sec7ΔC (WT and N653A), myristoylated Arf1, and ΔN17-Arf1 were purified from E. coli and resolved on a 15% SDS-PAGE gel. The asterisk indicates a contaminant. C, MALS coupled to gel filtration results for Sec7ΔC WT (left panel) and N653A mutant (right panel). Both constructs are dimeric.

Article Snippet: Plasmids and yeast strains used in this study are reported in and . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Name Description Vector backbone Source pET28 T7 promoter-driven expression plasmid Novagen pMR1 6 × His-Sec7ΔC (residues 203–1017) pET28 Ref. 14 pBCR563 6 × His-Sec7ΔC N653A (residues 203–1017) pET28 Ref. 15 pBCR389 6 × His-Sec7ΔC + HDS1 (residues 203–1220) pET28 Ref. 14 pSLH101 6 × His-Sec7ΔC + HDS1 N653A (residues 203–1220) pET28 This study pFastBacHT Baculovirus plasmid vector Invitrogen pBCR314 6 × His-Sec7f (residues 203–2009) pFastBacHT Ref. 14 2012 pSLH102 6 × His-Sec7f N653A (residues 203–2009) pFastBacHT This study pRS415 Centromeric LEU2 plasmid Ref. 44 pRS416 Centromeric URA3 plasmid Ref. 44 pCF1045 SEC7 (includes ∼1 kb of 5′-UTR) pRS415 Ref. 14 pCF1084 GFP-Sec7 driven by PSEC7 pRS415 Ref. 14 pBCR573 GFP-Sec7 N653A driven by PSEC7 pRS415 Ref. 15 pSLH103 GFP-Sec7 D655A driven by PSEC7 pRS415 This study pSLH104 GFP-Sec7 DC655AA driven by PSEC7 pRS415 This study pCF1140 GFP-Sec7ΔC (residues 1–1020) driven by PSEC7 pRS415 Ref. 14 pCF1141 GFP-Sec7ΔC + HDS1 (residues 1–1215) driven by PSEC7 pRS415 Ref. 14 pSLH105 GFP-Sec7ΔC + HDS1 N653A (residues 1–1215) driven by PSEC7 pRS415 This study pRS425 Yeast 2-μm vector with LEU2 marker pRS425 Ref. 44 pCF1271 SEC7 gene in pRS425 pRS425 pSLH106 SEC7 N653A gene in pRS425 pRS425 This study pNmt1 Nmt1 ( S. cerevisiae ) in pCYC plasmid Ref. 45 pArf1 Arf1 (S. cerevisiae) pET3c Ref. 46 pCF1053 6 × His-Arf1Δ N17 pET28 Ref. 14 Open in a separate window Plasmids used in this study table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Name Genotype Source SEY6210.1 MATa his3-200 leu2-3,112 lys2-801 trp1-901 ura3-52 suc2-9 Ref. 42 CFY409 BY4742 sec7 Δ:: KANMX +pCF1043 Ref. 14 CFY863 CFY409 arf1 Δ:: HIS3 Ref. 14 Open in a separate window Yeast strains used in this study

Techniques: Mutagenesis, Construct, Recombinant, Purification, SDS Page, Staining, Filtration

N653A mutation hinders GEF activity on membranes. A, representative nucleotide exchange (GEF activity) data for the activation of myr–Arf1 by different Sec7 constructs in the presence of liposome membranes. The intrinsic tryptophan fluorescence of Arf1 increases upon GTP-binding. B, quantified rates of nucleotide exchange of myr–Arf1 by WT and N653A Sec7ΔC+HDS1 and Sec7ΔC constructs in the presence of membranes. Quantification was performed from reaction rates (n = 3) from curves that were fit to a single exponential and normalized for measured GEF concentration to obtain the overall reaction rate. Points indicate actual data values. The error bars represent 95% confidence intervals; significance was measured by one-way ANOVA with Tukey's test for multiple comparisons. ***, p < 0.001; *, p < 0.05; n.s., not significant. C, liposome flotation assays showing membrane recruitment of WT Sec7ΔC+HDS1, but not N653A Sec7ΔC+HDS1, by activated myr–Arf1–GTP. D, liposome flotation assays showing recruitment of both WT and N653A Sec7f to membranes by activated myr–Arf1–GTP.

Journal: The Journal of Biological Chemistry

Article Title: The HUS box is required for allosteric regulation of the Sec7 Arf-GEF

doi: 10.1074/jbc.RA117.001318

Figure Lengend Snippet: N653A mutation hinders GEF activity on membranes. A, representative nucleotide exchange (GEF activity) data for the activation of myr–Arf1 by different Sec7 constructs in the presence of liposome membranes. The intrinsic tryptophan fluorescence of Arf1 increases upon GTP-binding. B, quantified rates of nucleotide exchange of myr–Arf1 by WT and N653A Sec7ΔC+HDS1 and Sec7ΔC constructs in the presence of membranes. Quantification was performed from reaction rates (n = 3) from curves that were fit to a single exponential and normalized for measured GEF concentration to obtain the overall reaction rate. Points indicate actual data values. The error bars represent 95% confidence intervals; significance was measured by one-way ANOVA with Tukey's test for multiple comparisons. ***, p < 0.001; *, p < 0.05; n.s., not significant. C, liposome flotation assays showing membrane recruitment of WT Sec7ΔC+HDS1, but not N653A Sec7ΔC+HDS1, by activated myr–Arf1–GTP. D, liposome flotation assays showing recruitment of both WT and N653A Sec7f to membranes by activated myr–Arf1–GTP.

Article Snippet: Plasmids and yeast strains used in this study are reported in and . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Name Description Vector backbone Source pET28 T7 promoter-driven expression plasmid Novagen pMR1 6 × His-Sec7ΔC (residues 203–1017) pET28 Ref. 14 pBCR563 6 × His-Sec7ΔC N653A (residues 203–1017) pET28 Ref. 15 pBCR389 6 × His-Sec7ΔC + HDS1 (residues 203–1220) pET28 Ref. 14 pSLH101 6 × His-Sec7ΔC + HDS1 N653A (residues 203–1220) pET28 This study pFastBacHT Baculovirus plasmid vector Invitrogen pBCR314 6 × His-Sec7f (residues 203–2009) pFastBacHT Ref. 14 2012 pSLH102 6 × His-Sec7f N653A (residues 203–2009) pFastBacHT This study pRS415 Centromeric LEU2 plasmid Ref. 44 pRS416 Centromeric URA3 plasmid Ref. 44 pCF1045 SEC7 (includes ∼1 kb of 5′-UTR) pRS415 Ref. 14 pCF1084 GFP-Sec7 driven by PSEC7 pRS415 Ref. 14 pBCR573 GFP-Sec7 N653A driven by PSEC7 pRS415 Ref. 15 pSLH103 GFP-Sec7 D655A driven by PSEC7 pRS415 This study pSLH104 GFP-Sec7 DC655AA driven by PSEC7 pRS415 This study pCF1140 GFP-Sec7ΔC (residues 1–1020) driven by PSEC7 pRS415 Ref. 14 pCF1141 GFP-Sec7ΔC + HDS1 (residues 1–1215) driven by PSEC7 pRS415 Ref. 14 pSLH105 GFP-Sec7ΔC + HDS1 N653A (residues 1–1215) driven by PSEC7 pRS415 This study pRS425 Yeast 2-μm vector with LEU2 marker pRS425 Ref. 44 pCF1271 SEC7 gene in pRS425 pRS425 pSLH106 SEC7 N653A gene in pRS425 pRS425 This study pNmt1 Nmt1 ( S. cerevisiae ) in pCYC plasmid Ref. 45 pArf1 Arf1 (S. cerevisiae) pET3c Ref. 46 pCF1053 6 × His-Arf1Δ N17 pET28 Ref. 14 Open in a separate window Plasmids used in this study table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Name Genotype Source SEY6210.1 MATa his3-200 leu2-3,112 lys2-801 trp1-901 ura3-52 suc2-9 Ref. 42 CFY409 BY4742 sec7 Δ:: KANMX +pCF1043 Ref. 14 CFY863 CFY409 arf1 Δ:: HIS3 Ref. 14 Open in a separate window Yeast strains used in this study

Techniques: Mutagenesis, Activity Assay, Activation Assay, Construct, Fluorescence, Binding Assay, Concentration Assay

The N653A mutation disrupts localization of Sec7ΔC+HDS1. A, localization of an extra copy of GFP-tagged Sec7 constructs in live yeast cells. Constructs were expressed under the endogenous SEC7 promoter on centromeric plasmids in WT cells (CFY188) and imaged in log phase. Dashed circles indicate cell boundaries. Scale bar, 2 μm. DIC, differential interference contrast. See also Fig. S1. B, localization of a single copy of GFP-tagged WT and mutant full-length Sec7 constructs expressed under the endogenous promoter on centromeric plasmids after shuffling. Plasmids were introduced into the sec7Δ shuffling strain (CFY409) and then plated on 5-fluoroorotic acid (5-FOA). Live cells were then imaged in log phase. Dashed circles indicate cells. Scale bar, 2 μm.

Journal: The Journal of Biological Chemistry

Article Title: The HUS box is required for allosteric regulation of the Sec7 Arf-GEF

doi: 10.1074/jbc.RA117.001318

Figure Lengend Snippet: The N653A mutation disrupts localization of Sec7ΔC+HDS1. A, localization of an extra copy of GFP-tagged Sec7 constructs in live yeast cells. Constructs were expressed under the endogenous SEC7 promoter on centromeric plasmids in WT cells (CFY188) and imaged in log phase. Dashed circles indicate cell boundaries. Scale bar, 2 μm. DIC, differential interference contrast. See also Fig. S1. B, localization of a single copy of GFP-tagged WT and mutant full-length Sec7 constructs expressed under the endogenous promoter on centromeric plasmids after shuffling. Plasmids were introduced into the sec7Δ shuffling strain (CFY409) and then plated on 5-fluoroorotic acid (5-FOA). Live cells were then imaged in log phase. Dashed circles indicate cells. Scale bar, 2 μm.

Article Snippet: Plasmids and yeast strains used in this study are reported in and . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Name Description Vector backbone Source pET28 T7 promoter-driven expression plasmid Novagen pMR1 6 × His-Sec7ΔC (residues 203–1017) pET28 Ref. 14 pBCR563 6 × His-Sec7ΔC N653A (residues 203–1017) pET28 Ref. 15 pBCR389 6 × His-Sec7ΔC + HDS1 (residues 203–1220) pET28 Ref. 14 pSLH101 6 × His-Sec7ΔC + HDS1 N653A (residues 203–1220) pET28 This study pFastBacHT Baculovirus plasmid vector Invitrogen pBCR314 6 × His-Sec7f (residues 203–2009) pFastBacHT Ref. 14 2012 pSLH102 6 × His-Sec7f N653A (residues 203–2009) pFastBacHT This study pRS415 Centromeric LEU2 plasmid Ref. 44 pRS416 Centromeric URA3 plasmid Ref. 44 pCF1045 SEC7 (includes ∼1 kb of 5′-UTR) pRS415 Ref. 14 pCF1084 GFP-Sec7 driven by PSEC7 pRS415 Ref. 14 pBCR573 GFP-Sec7 N653A driven by PSEC7 pRS415 Ref. 15 pSLH103 GFP-Sec7 D655A driven by PSEC7 pRS415 This study pSLH104 GFP-Sec7 DC655AA driven by PSEC7 pRS415 This study pCF1140 GFP-Sec7ΔC (residues 1–1020) driven by PSEC7 pRS415 Ref. 14 pCF1141 GFP-Sec7ΔC + HDS1 (residues 1–1215) driven by PSEC7 pRS415 Ref. 14 pSLH105 GFP-Sec7ΔC + HDS1 N653A (residues 1–1215) driven by PSEC7 pRS415 This study pRS425 Yeast 2-μm vector with LEU2 marker pRS425 Ref. 44 pCF1271 SEC7 gene in pRS425 pRS425 pSLH106 SEC7 N653A gene in pRS425 pRS425 This study pNmt1 Nmt1 ( S. cerevisiae ) in pCYC plasmid Ref. 45 pArf1 Arf1 (S. cerevisiae) pET3c Ref. 46 pCF1053 6 × His-Arf1Δ N17 pET28 Ref. 14 Open in a separate window Plasmids used in this study table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Name Genotype Source SEY6210.1 MATa his3-200 leu2-3,112 lys2-801 trp1-901 ura3-52 suc2-9 Ref. 42 CFY409 BY4742 sec7 Δ:: KANMX +pCF1043 Ref. 14 CFY863 CFY409 arf1 Δ:: HIS3 Ref. 14 Open in a separate window Yeast strains used in this study

Techniques: Mutagenesis, Construct

Plasmids used in this study

Journal: The Journal of Biological Chemistry

Article Title: The HUS box is required for allosteric regulation of the Sec7 Arf-GEF

doi: 10.1074/jbc.RA117.001318

Figure Lengend Snippet: Plasmids used in this study

Article Snippet: Plasmids and yeast strains used in this study are reported in and . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Name Description Vector backbone Source pET28 T7 promoter-driven expression plasmid Novagen pMR1 6 × His-Sec7ΔC (residues 203–1017) pET28 Ref. 14 pBCR563 6 × His-Sec7ΔC N653A (residues 203–1017) pET28 Ref. 15 pBCR389 6 × His-Sec7ΔC + HDS1 (residues 203–1220) pET28 Ref. 14 pSLH101 6 × His-Sec7ΔC + HDS1 N653A (residues 203–1220) pET28 This study pFastBacHT Baculovirus plasmid vector Invitrogen pBCR314 6 × His-Sec7f (residues 203–2009) pFastBacHT Ref. 14 2012 pSLH102 6 × His-Sec7f N653A (residues 203–2009) pFastBacHT This study pRS415 Centromeric LEU2 plasmid Ref. 44 pRS416 Centromeric URA3 plasmid Ref. 44 pCF1045 SEC7 (includes ∼1 kb of 5′-UTR) pRS415 Ref. 14 pCF1084 GFP-Sec7 driven by PSEC7 pRS415 Ref. 14 pBCR573 GFP-Sec7 N653A driven by PSEC7 pRS415 Ref. 15 pSLH103 GFP-Sec7 D655A driven by PSEC7 pRS415 This study pSLH104 GFP-Sec7 DC655AA driven by PSEC7 pRS415 This study pCF1140 GFP-Sec7ΔC (residues 1–1020) driven by PSEC7 pRS415 Ref. 14 pCF1141 GFP-Sec7ΔC + HDS1 (residues 1–1215) driven by PSEC7 pRS415 Ref. 14 pSLH105 GFP-Sec7ΔC + HDS1 N653A (residues 1–1215) driven by PSEC7 pRS415 This study pRS425 Yeast 2-μm vector with LEU2 marker pRS425 Ref. 44 pCF1271 SEC7 gene in pRS425 pRS425 pSLH106 SEC7 N653A gene in pRS425 pRS425 This study pNmt1 Nmt1 ( S. cerevisiae ) in pCYC plasmid Ref. 45 pArf1 Arf1 (S. cerevisiae) pET3c Ref. 46 pCF1053 6 × His-Arf1Δ N17 pET28 Ref. 14 Open in a separate window Plasmids used in this study table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Name Genotype Source SEY6210.1 MATa his3-200 leu2-3,112 lys2-801 trp1-901 ura3-52 suc2-9 Ref. 42 CFY409 BY4742 sec7 Δ:: KANMX +pCF1043 Ref. 14 CFY863 CFY409 arf1 Δ:: HIS3 Ref. 14 Open in a separate window Yeast strains used in this study

Techniques: Plasmid Preparation, Expressing, Marker