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human itag5 cdna  (Addgene inc)


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    Addgene inc human itag5 cdna
    ( 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 <t>Itag5</t> (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. .
    Human Itag5 Cdna, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/human+itag5+cdna/Alpha+5+integrin-GFP+(Plasmid+%2315238)/pmc11624278-459-29-37
    Average 93 stars, based on 29 article reviews
    human itag5 cdna - by Bioz Stars, 2026-09
    93/100 stars

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    1) Product Images from "The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation"

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

    Journal: EMBO Reports

    doi: 10.1038/s44319-024-00300-9

    ( 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. .
    Figure Legend 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. .

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

    Related Articles

    Generated:

    Article Title: The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation
    Article Snippet: The Itgb1 and Itga5 cDNAs carrying lysine for arginine substitutions in the cytoplasmic domain (Itgb1 8xKR and Itga5 4xKR ) were previously generated by site-directed mutagenesis (Böttcher et al, ) and cloned into the HSC1 retroviral vector (a gift from James Ellis, Addgene plasmid # 58254). .. 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. .. The cDNAs encoding Ub-WT (a gift from Ted Dawson, Addgene plasmid # 17608), Ub-K48R (a gift from Ted Dawson, Addgene plasmid # 17604), and Ub-K63R (a gift from Cecile Pickart, Addgene plasmid # 18898) were cloned into pEGFP-N1 vector (Clontech).

    Article Title: The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation.
    Article Snippet: The functions of integrins are tightly regulated via multiple mechanisms including trafficking and degradation.. Integrins are repeatedly internalized, routed into the endosomal system and either degraded by the lysosome or recycled back to the plasma membrane.. The ubiquitin system dictates whether internalized proteins are degraded or recycled.

    Article Title: The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation
    Article Snippet: The Itgb1 and Itga5 cDNAs carrying lysine for arginine substitutions in the cytoplasmic domain (Itgb1 8xKR and Itga5 4xKR ) were previously generated by site-directed mutagenesis ( Böttcher et al ., 2012 ) and cloned into the HSC1 retroviral vector (a gift from James Ellis, Addgene plasmid # 58254). .. 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. .. The cDNAs encoding Ub-WT (a gift from Ted Dawson, Addgene plasmid # 17608), Ub-K48R (a gift from Ted Dawson, Addgene plasmid # 17604) and Ub-K63R (a gift from Cecile Pickart, Addgene plasmid # 18898) were cloned into pEGFP-N1 vector (Clontech).

    Plasmid Preparation:

    Article Title: The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation
    Article Snippet: The Itgb1 and Itga5 cDNAs carrying lysine for arginine substitutions in the cytoplasmic domain (Itgb1 8xKR and Itga5 4xKR ) were previously generated by site-directed mutagenesis (Böttcher et al, ) and cloned into the HSC1 retroviral vector (a gift from James Ellis, Addgene plasmid # 58254). .. 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. .. The cDNAs encoding Ub-WT (a gift from Ted Dawson, Addgene plasmid # 17608), Ub-K48R (a gift from Ted Dawson, Addgene plasmid # 17604), and Ub-K63R (a gift from Cecile Pickart, Addgene plasmid # 18898) were cloned into pEGFP-N1 vector (Clontech).

    Article Title: The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation.
    Article Snippet: The functions of integrins are tightly regulated via multiple mechanisms including trafficking and degradation.. Integrins are repeatedly internalized, routed into the endosomal system and either degraded by the lysosome or recycled back to the plasma membrane.. The ubiquitin system dictates whether internalized proteins are degraded or recycled.

    Article Title: The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation
    Article Snippet: The Itgb1 and Itga5 cDNAs carrying lysine for arginine substitutions in the cytoplasmic domain (Itgb1 8xKR and Itga5 4xKR ) were previously generated by site-directed mutagenesis ( Böttcher et al ., 2012 ) and cloned into the HSC1 retroviral vector (a gift from James Ellis, Addgene plasmid # 58254). .. 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. .. The cDNAs encoding Ub-WT (a gift from Ted Dawson, Addgene plasmid # 17608), Ub-K48R (a gift from Ted Dawson, Addgene plasmid # 17604) and Ub-K63R (a gift from Cecile Pickart, Addgene plasmid # 18898) were cloned into pEGFP-N1 vector (Clontech).

    Clone Assay:

    Article Title: The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation
    Article Snippet: The Itgb1 and Itga5 cDNAs carrying lysine for arginine substitutions in the cytoplasmic domain (Itgb1 8xKR and Itga5 4xKR ) were previously generated by site-directed mutagenesis (Böttcher et al, ) and cloned into the HSC1 retroviral vector (a gift from James Ellis, Addgene plasmid # 58254). .. 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. .. The cDNAs encoding Ub-WT (a gift from Ted Dawson, Addgene plasmid # 17608), Ub-K48R (a gift from Ted Dawson, Addgene plasmid # 17604), and Ub-K63R (a gift from Cecile Pickart, Addgene plasmid # 18898) were cloned into pEGFP-N1 vector (Clontech).

    Article Title: The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation.
    Article Snippet: The functions of integrins are tightly regulated via multiple mechanisms including trafficking and degradation.. Integrins are repeatedly internalized, routed into the endosomal system and either degraded by the lysosome or recycled back to the plasma membrane.. The ubiquitin system dictates whether internalized proteins are degraded or recycled.

    Article Title: The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation
    Article Snippet: The Itgb1 and Itga5 cDNAs carrying lysine for arginine substitutions in the cytoplasmic domain (Itgb1 8xKR and Itga5 4xKR ) were previously generated by site-directed mutagenesis ( Böttcher et al ., 2012 ) and cloned into the HSC1 retroviral vector (a gift from James Ellis, Addgene plasmid # 58254). .. 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. .. The cDNAs encoding Ub-WT (a gift from Ted Dawson, Addgene plasmid # 17608), Ub-K48R (a gift from Ted Dawson, Addgene plasmid # 17604) and Ub-K63R (a gift from Cecile Pickart, Addgene plasmid # 18898) were cloned into pEGFP-N1 vector (Clontech).



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    human itag5 cdna  (Addgene inc)
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    Addgene inc human itag5 cdna
    ( 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 <t>Itag5</t> (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. .
    Human Itag5 Cdna, 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/product/human+itag5+cdna/Alpha+5+integrin-GFP+(Plasmid+%2315238)/pmc11624278-459-29-37
    Average 93 stars, based on 1 article reviews
    human itag5 cdna - by Bioz Stars, 2026-09
    93/100 stars
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    ( 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