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    Addgene inc flag otud5
    <t>OTUD5</t> is a specific stabilizer of UBR5. ( A ) Indicated siRNAs (20 nM) were transfected to 293T cells, pellets were harvested after 72 hours and UBR5 levels were detected by western blots. Band intensities were internally normalized to tubulin and quantified using Image J. ( B ) siRNAs were transfected to HeLa, followed by Cycloheximide treatment (10 µM, for indicated hours) and western blotting. ( C ) UBR5 foci formation was induced by UVC through 3 µm micropore filter (100J/m 2 , 1 hour recovery) following the siRNA transfections ( n = 20 each). See ‘Materials and Method’ section for RFI description. Bottom panel is for testing various (OTU DUB members) siRNAs for UBR5 foci formation ( n = 20 each, **** indicates P -value < 0.0005). ( D ) Confirmation of OTUD5 CRISPR KO HeLa clones (#1 is CRISPR-Cas9 clone, #2 is Double-Nickase clone). ( E ) OTUD5 KO#1 cells were transfected with WT or C224S OTUD5 plasmids, and analyzed by western blotting. ( F ) 293T cells were transfected with indicated siRNAs and the plasmids, harvested pellets were lysed and anti-FLAG IP assay was performed. Bands above unmodified UBR5 are increased in OTUD5 Knockdown cells. ( G ) Cyclohexamide chase analysis (10 µM, for indicated hours) in OTUD5 KO cells complemented with either WT or catalytically inactive C2768A FLAG-UBR5 plasmids. ( H ) 293T cells were transfected with 3xFLAG-OTUD5 plasmid and anti-FLAG IP was performed. ( I ) Purified recombinant OTUD5 and 6xHis-UBR5 proteins were mixed, and the mixture was subject to Ni beads pulldown. In Ni 2+ -only, OTUD5 proteins were added without UBR5. Shown is the coomassie stained gel.
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    OTUD5 is a specific stabilizer of UBR5. ( A ) Indicated siRNAs (20 nM) were transfected to 293T cells, pellets were harvested after 72 hours and UBR5 levels were detected by western blots. Band intensities were internally normalized to tubulin and quantified using Image J. ( B ) siRNAs were transfected to HeLa, followed by Cycloheximide treatment (10 µM, for indicated hours) and western blotting. ( C ) UBR5 foci formation was induced by UVC through 3 µm micropore filter (100J/m 2 , 1 hour recovery) following the siRNA transfections ( n = 20 each). See ‘Materials and Method’ section for RFI description. Bottom panel is for testing various (OTU DUB members) siRNAs for UBR5 foci formation ( n = 20 each, **** indicates P -value < 0.0005). ( D ) Confirmation of OTUD5 CRISPR KO HeLa clones (#1 is CRISPR-Cas9 clone, #2 is Double-Nickase clone). ( E ) OTUD5 KO#1 cells were transfected with WT or C224S OTUD5 plasmids, and analyzed by western blotting. ( F ) 293T cells were transfected with indicated siRNAs and the plasmids, harvested pellets were lysed and anti-FLAG IP assay was performed. Bands above unmodified UBR5 are increased in OTUD5 Knockdown cells. ( G ) Cyclohexamide chase analysis (10 µM, for indicated hours) in OTUD5 KO cells complemented with either WT or catalytically inactive C2768A FLAG-UBR5 plasmids. ( H ) 293T cells were transfected with 3xFLAG-OTUD5 plasmid and anti-FLAG IP was performed. ( I ) Purified recombinant OTUD5 and 6xHis-UBR5 proteins were mixed, and the mixture was subject to Ni beads pulldown. In Ni 2+ -only, OTUD5 proteins were added without UBR5. Shown is the coomassie stained gel.

    Journal: Nucleic Acids Research

    Article Title: The OTUD5–UBR5 complex regulates FACT-mediated transcription at damaged chromatin

    doi: 10.1093/nar/gky1219

    Figure Lengend Snippet: OTUD5 is a specific stabilizer of UBR5. ( A ) Indicated siRNAs (20 nM) were transfected to 293T cells, pellets were harvested after 72 hours and UBR5 levels were detected by western blots. Band intensities were internally normalized to tubulin and quantified using Image J. ( B ) siRNAs were transfected to HeLa, followed by Cycloheximide treatment (10 µM, for indicated hours) and western blotting. ( C ) UBR5 foci formation was induced by UVC through 3 µm micropore filter (100J/m 2 , 1 hour recovery) following the siRNA transfections ( n = 20 each). See ‘Materials and Method’ section for RFI description. Bottom panel is for testing various (OTU DUB members) siRNAs for UBR5 foci formation ( n = 20 each, **** indicates P -value < 0.0005). ( D ) Confirmation of OTUD5 CRISPR KO HeLa clones (#1 is CRISPR-Cas9 clone, #2 is Double-Nickase clone). ( E ) OTUD5 KO#1 cells were transfected with WT or C224S OTUD5 plasmids, and analyzed by western blotting. ( F ) 293T cells were transfected with indicated siRNAs and the plasmids, harvested pellets were lysed and anti-FLAG IP assay was performed. Bands above unmodified UBR5 are increased in OTUD5 Knockdown cells. ( G ) Cyclohexamide chase analysis (10 µM, for indicated hours) in OTUD5 KO cells complemented with either WT or catalytically inactive C2768A FLAG-UBR5 plasmids. ( H ) 293T cells were transfected with 3xFLAG-OTUD5 plasmid and anti-FLAG IP was performed. ( I ) Purified recombinant OTUD5 and 6xHis-UBR5 proteins were mixed, and the mixture was subject to Ni beads pulldown. In Ni 2+ -only, OTUD5 proteins were added without UBR5. Shown is the coomassie stained gel.

    Article Snippet: The U2OS cells stably expressing SNAP-H2A were generated through G418 selection and clonal isolation. pDEST_Tet_CMV_FLAG-OTUD5 was a gift from Wade Harper (Addgene plasmid # 22610) ( ) and was used for generating HeLa cells stably expressing FLAG-OTUD5. pDEST10-6xHis-UBR5 plasmid was a gift from Sally Kornbluth ( ) and was used for recombinant protein production from SF9 cells.

    Techniques: Transfection, Western Blot, CRISPR, Clone Assay, Plasmid Preparation, Purification, Recombinant, Staining

    OTUD5 localizes to UV and DSB-induced chromatin lesions. ( A ) HeLa cells were irradiated with UVC (100J/m 2 ) through 3 µm micropore filters, and 1 hour later cells were fixed and co-stained with anti-γH2AX and OTUD5 antibodies. ( B ) HeLa cells were transfected with GFP-OTUD5 plasmid, irradiated with UVC through micropore filter as in A, and the fixed cells were stained for γH2AX. ( C ) mCherry-LacI-Fok1 fusion proteins (expressed in PTuner 263 U2OS cells) were induced by Shield-1 and 4-OHT (see ‘Materials and Methods’ section), and the fixed cells were stained with endogenous OTUD5 antibody. ( D ) The PTuner U2OS reporter cells were transfected with GFP-OTUD5. ( E ) 293T cells treated with or without Bleomycin (5 µM, 16 hours) were subject to fractionation assay (S = NaCl 100 mM eluate, P = the rest of pellet containing chromatin fraction) and the eluates were analyzed by western blots. ( F ) HeLa cells stably expressing Dox-inducible FLAG-OTUD5 were treated with Tetracyclin (10 µg/ml), followed by UV (30 J/m 2 ) irradiation through micropore (3 µm) filter. PLA was performed with anti-FLAG and anti-UBR5 antibodies (see ‘Materials and Methods’ section). The slides were also co-stained with anti-53BP1 antibody to mark the DSB lesions. On the right is quantification for relative number of interactions per nucleus ( N = 17). Scale bars indicate 10 μm. (**** indicates P -value <0.0005). Experiments were done in triplicates.

    Journal: Nucleic Acids Research

    Article Title: The OTUD5–UBR5 complex regulates FACT-mediated transcription at damaged chromatin

    doi: 10.1093/nar/gky1219

    Figure Lengend Snippet: OTUD5 localizes to UV and DSB-induced chromatin lesions. ( A ) HeLa cells were irradiated with UVC (100J/m 2 ) through 3 µm micropore filters, and 1 hour later cells were fixed and co-stained with anti-γH2AX and OTUD5 antibodies. ( B ) HeLa cells were transfected with GFP-OTUD5 plasmid, irradiated with UVC through micropore filter as in A, and the fixed cells were stained for γH2AX. ( C ) mCherry-LacI-Fok1 fusion proteins (expressed in PTuner 263 U2OS cells) were induced by Shield-1 and 4-OHT (see ‘Materials and Methods’ section), and the fixed cells were stained with endogenous OTUD5 antibody. ( D ) The PTuner U2OS reporter cells were transfected with GFP-OTUD5. ( E ) 293T cells treated with or without Bleomycin (5 µM, 16 hours) were subject to fractionation assay (S = NaCl 100 mM eluate, P = the rest of pellet containing chromatin fraction) and the eluates were analyzed by western blots. ( F ) HeLa cells stably expressing Dox-inducible FLAG-OTUD5 were treated with Tetracyclin (10 µg/ml), followed by UV (30 J/m 2 ) irradiation through micropore (3 µm) filter. PLA was performed with anti-FLAG and anti-UBR5 antibodies (see ‘Materials and Methods’ section). The slides were also co-stained with anti-53BP1 antibody to mark the DSB lesions. On the right is quantification for relative number of interactions per nucleus ( N = 17). Scale bars indicate 10 μm. (**** indicates P -value <0.0005). Experiments were done in triplicates.

    Article Snippet: The U2OS cells stably expressing SNAP-H2A were generated through G418 selection and clonal isolation. pDEST_Tet_CMV_FLAG-OTUD5 was a gift from Wade Harper (Addgene plasmid # 22610) ( ) and was used for generating HeLa cells stably expressing FLAG-OTUD5. pDEST10-6xHis-UBR5 plasmid was a gift from Sally Kornbluth ( ) and was used for recombinant protein production from SF9 cells.

    Techniques: Irradiation, Staining, Transfection, Plasmid Preparation, Fractionation, Western Blot, Stable Transfection, Expressing

    OTUD5 regulates transcription repression at damaged chromatin. ( A ) Ptuner 263 cells transfected with indicated siRNAs were treated with Shield-1/4-OHT and Tetracyclin for inducing LacI-mCherry-Fok1 fusion nuclease and YFP-MS2 reporter, respectively, 3 hours prior to fixing. The representative images are shown. ( B ) As in A, the assays were performed with indicated siRNAs and the YFP-MS2 RFI at Fok1 sites were quantified and plotted. See ‘Materials and Methods’ section for RFI description. The assay was performed in triplicates ( N = 35 each, **** indicates P -value < 0.0005). Scale bars indicate 10 μm. For C and E, the UBR5 and OTUD5 CRISPR KO HeLa cells were treated with Bleomycin (5 µM) for 16 hours, fixed, then co-stained with γH2AX and Pol II P-Ser2 ( C ), or γH2AX and 5-EU ( E ). On the right are the quantification of the γH2AX overlap with Pol II (P-Ser) or 5-EU in WT and KO cells. The Pearson’s correlation was measured using the ‘co-localization’ finder plug-in in Image J. The assay was performed in triplicates ( N = 50 each). A score of ‘1’ indicates 100% correlation between red and green pixels; a score of ‘−1’ indicates inverse correlation. ‘0’ indicates no relationship. Similar experiments were done using siRNA transfected cells for the PolII-γH2AX overlap analysis ( D ) and the 5EU-γH2AX overlap analysis ( F ). Representative images for these assays are shown in , respectively). All analysis were done in triplicates. (**** indicates P -value < 0.0005).

    Journal: Nucleic Acids Research

    Article Title: The OTUD5–UBR5 complex regulates FACT-mediated transcription at damaged chromatin

    doi: 10.1093/nar/gky1219

    Figure Lengend Snippet: OTUD5 regulates transcription repression at damaged chromatin. ( A ) Ptuner 263 cells transfected with indicated siRNAs were treated with Shield-1/4-OHT and Tetracyclin for inducing LacI-mCherry-Fok1 fusion nuclease and YFP-MS2 reporter, respectively, 3 hours prior to fixing. The representative images are shown. ( B ) As in A, the assays were performed with indicated siRNAs and the YFP-MS2 RFI at Fok1 sites were quantified and plotted. See ‘Materials and Methods’ section for RFI description. The assay was performed in triplicates ( N = 35 each, **** indicates P -value < 0.0005). Scale bars indicate 10 μm. For C and E, the UBR5 and OTUD5 CRISPR KO HeLa cells were treated with Bleomycin (5 µM) for 16 hours, fixed, then co-stained with γH2AX and Pol II P-Ser2 ( C ), or γH2AX and 5-EU ( E ). On the right are the quantification of the γH2AX overlap with Pol II (P-Ser) or 5-EU in WT and KO cells. The Pearson’s correlation was measured using the ‘co-localization’ finder plug-in in Image J. The assay was performed in triplicates ( N = 50 each). A score of ‘1’ indicates 100% correlation between red and green pixels; a score of ‘−1’ indicates inverse correlation. ‘0’ indicates no relationship. Similar experiments were done using siRNA transfected cells for the PolII-γH2AX overlap analysis ( D ) and the 5EU-γH2AX overlap analysis ( F ). Representative images for these assays are shown in , respectively). All analysis were done in triplicates. (**** indicates P -value < 0.0005).

    Article Snippet: The U2OS cells stably expressing SNAP-H2A were generated through G418 selection and clonal isolation. pDEST_Tet_CMV_FLAG-OTUD5 was a gift from Wade Harper (Addgene plasmid # 22610) ( ) and was used for generating HeLa cells stably expressing FLAG-OTUD5. pDEST10-6xHis-UBR5 plasmid was a gift from Sally Kornbluth ( ) and was used for recombinant protein production from SF9 cells.

    Techniques: Transfection, CRISPR, Staining

    OTUD5 interacts with SPT16 and regulates the dynamics of SPT16 recruitment at damaged sites. ( A ) Mass spectrometry analysis of FLAG-OTUD5 IP in HeLa cells (experiment #1) or GST-OTUD5 pulldown with 293T lysate (experiment #2). Key reproducible peptides are shown. ( B ) 293T lysate were subject to anti-SPT16 IP, with or without pre-treatment of the lysate with benzonase for 1 hour at 37C (U = units), then the eluate was analyzed. ( C ). 293T cells were subject to anti - OTUD5 IP and the eluate was analyzed. ( D ) Ptuner 263 cells were treated with Shield1-4OHT to induce Fok1, and the cells were fixed and stained with SPT16 (top) or OTUD5 (bottom) antibodies. ( E ) SPT16-OTUD5 PLA was performed in HeLa cells (Top) and quantification is shown. The assay was performed in triplicates ( N = 20). ( F ) PLA (anti-UBR5 + anti-SPT16 antibodies) was performed in HeLa cells transfected with either siControl or siOTUD5. Quantification of number of PLA signals per nucleus is shown. The assay was performed in triplicates ( N = 20). ( G ) Ptuner cells transfected with indicated siRNAs were treated with Shield1/4OHT and Tetracyclin to induce Fok1 and YFP-MS2, respectively. The assay was performed in triplicates ( N = 30 each) and the YFP-MS2 RFI was quantified on the right. All the scale bars indicate 10 μm. (**** indicates P -value < 0.0005, *** <0.005, N.S. = not significant).

    Journal: Nucleic Acids Research

    Article Title: The OTUD5–UBR5 complex regulates FACT-mediated transcription at damaged chromatin

    doi: 10.1093/nar/gky1219

    Figure Lengend Snippet: OTUD5 interacts with SPT16 and regulates the dynamics of SPT16 recruitment at damaged sites. ( A ) Mass spectrometry analysis of FLAG-OTUD5 IP in HeLa cells (experiment #1) or GST-OTUD5 pulldown with 293T lysate (experiment #2). Key reproducible peptides are shown. ( B ) 293T lysate were subject to anti-SPT16 IP, with or without pre-treatment of the lysate with benzonase for 1 hour at 37C (U = units), then the eluate was analyzed. ( C ). 293T cells were subject to anti - OTUD5 IP and the eluate was analyzed. ( D ) Ptuner 263 cells were treated with Shield1-4OHT to induce Fok1, and the cells were fixed and stained with SPT16 (top) or OTUD5 (bottom) antibodies. ( E ) SPT16-OTUD5 PLA was performed in HeLa cells (Top) and quantification is shown. The assay was performed in triplicates ( N = 20). ( F ) PLA (anti-UBR5 + anti-SPT16 antibodies) was performed in HeLa cells transfected with either siControl or siOTUD5. Quantification of number of PLA signals per nucleus is shown. The assay was performed in triplicates ( N = 20). ( G ) Ptuner cells transfected with indicated siRNAs were treated with Shield1/4OHT and Tetracyclin to induce Fok1 and YFP-MS2, respectively. The assay was performed in triplicates ( N = 30 each) and the YFP-MS2 RFI was quantified on the right. All the scale bars indicate 10 μm. (**** indicates P -value < 0.0005, *** <0.005, N.S. = not significant).

    Article Snippet: The U2OS cells stably expressing SNAP-H2A were generated through G418 selection and clonal isolation. pDEST_Tet_CMV_FLAG-OTUD5 was a gift from Wade Harper (Addgene plasmid # 22610) ( ) and was used for generating HeLa cells stably expressing FLAG-OTUD5. pDEST10-6xHis-UBR5 plasmid was a gift from Sally Kornbluth ( ) and was used for recombinant protein production from SF9 cells.

    Techniques: Mass Spectrometry, Staining, Transfection

    OTUD5 and UBR5 regulates the deposition of new Histone H2A at the damage sites. ( A ) Enlargement of SPT16 foci in UBR5 and OTUD5 knockdown cells. HeLa cells were irradiated with UVC (100 J/m 2 ) through 3 μm micropore filters and fixed 1 hour after. The number of pixels in each γH2AX and SPT16 co-localized foci was measured using Image J, with each pixel represents an area of 0.2 μm 2 . The ratio of SPT16 to γH2AX was calculated by dividing the SPT16 area by the γH2AX area. The assay was performed in triplicates ( N = 35). Red bars indicate the median value for each set. ( B ) Ratio of SPT16 to Fok1 area in pTuner263 cells transfected with the siRNAs were quantified as in ( A ) The assay was performed in triplicates, N = 25. ( C ) Schematic for SNAP-H2A labeling and nucleosome integration. ( D ) U2OS cells stably expressing SNAP-H2A were first blocked with TMR Block and then irradiated with UVC (100 J/m 2 ) through 3 μm micropore filters. Cells were labeled with TMR Star (red) and then fixed at indicated time points. Cells were counter-stained for γH2AX. The assay was performed in triplicates. Vector quantification of RFI of TMR signal (SNAP-H2A) ( N = 20); see ‘Materials and Methods’ section for details. Scale bars indicate 10 μm. (**** indicates P -value < 0.0005).

    Journal: Nucleic Acids Research

    Article Title: The OTUD5–UBR5 complex regulates FACT-mediated transcription at damaged chromatin

    doi: 10.1093/nar/gky1219

    Figure Lengend Snippet: OTUD5 and UBR5 regulates the deposition of new Histone H2A at the damage sites. ( A ) Enlargement of SPT16 foci in UBR5 and OTUD5 knockdown cells. HeLa cells were irradiated with UVC (100 J/m 2 ) through 3 μm micropore filters and fixed 1 hour after. The number of pixels in each γH2AX and SPT16 co-localized foci was measured using Image J, with each pixel represents an area of 0.2 μm 2 . The ratio of SPT16 to γH2AX was calculated by dividing the SPT16 area by the γH2AX area. The assay was performed in triplicates ( N = 35). Red bars indicate the median value for each set. ( B ) Ratio of SPT16 to Fok1 area in pTuner263 cells transfected with the siRNAs were quantified as in ( A ) The assay was performed in triplicates, N = 25. ( C ) Schematic for SNAP-H2A labeling and nucleosome integration. ( D ) U2OS cells stably expressing SNAP-H2A were first blocked with TMR Block and then irradiated with UVC (100 J/m 2 ) through 3 μm micropore filters. Cells were labeled with TMR Star (red) and then fixed at indicated time points. Cells were counter-stained for γH2AX. The assay was performed in triplicates. Vector quantification of RFI of TMR signal (SNAP-H2A) ( N = 20); see ‘Materials and Methods’ section for details. Scale bars indicate 10 μm. (**** indicates P -value < 0.0005).

    Article Snippet: The U2OS cells stably expressing SNAP-H2A were generated through G418 selection and clonal isolation. pDEST_Tet_CMV_FLAG-OTUD5 was a gift from Wade Harper (Addgene plasmid # 22610) ( ) and was used for generating HeLa cells stably expressing FLAG-OTUD5. pDEST10-6xHis-UBR5 plasmid was a gift from Sally Kornbluth ( ) and was used for recombinant protein production from SF9 cells.

    Techniques: Irradiation, Transfection, Labeling, Stable Transfection, Expressing, Blocking Assay, Staining, Plasmid Preparation

    OTUD5 interacts with UBR5 and SPT16 through distinct regions. ( A ) PONDR software predicts the disordered regions within OTUD5 protein. Below is a schematic for the OTUD5 truncation plasmids used for interaction and other functional analyses. The summary of the assay results are shown on the right: ‘Binding’ was measured by co-IP assays as shown in ( B ), and ‘Stabilization’ was measured by western blots as shown in ( C ). ‘NLS’ is indicated on the schematics based on our analysis in . ( B ) 293T cells transfected with the indicated plasmids, then anti-FLAG IP was performed and the eluates were analyzed by western blotting ( C ). HeLa OTUD5 KO (clone#2) cells were transfected with indicated plasmids and UBR5 protein was analyzed. ( D ) Cross-species alignment on the N-terminus of OTUD5. ( E ) Purified WT or ΔUIM GST-OTUD5 recombinant proteins was mixed with 293T cell lysate, pulled down with glutathione beads, then the eluates were analyzed by western blots. ( F ) Ptuner263 cells were transfected with indicated OTUD5 truncates (EV = empty vector) then the Fok1 nucleases were induced by Shield1 and 4-OHT. Cells were fixed then stained with anti-FLAG antibody. The experiments were done in triplicates and on the right is the statistical information ( N = 30 each). ( G ) HeLa cells were transfected with indicated pBABE-FLAG-OTUD5 plasmids (NT = non transfected, EV = empty vector) and the PLA assay with anti-UBR5 and anti-FLAG (OTUD5) was performed ( N = 30). Scale bars indicate 10 μm. (**** indicates P -value < 0.0005, N.S. = not significant).

    Journal: Nucleic Acids Research

    Article Title: The OTUD5–UBR5 complex regulates FACT-mediated transcription at damaged chromatin

    doi: 10.1093/nar/gky1219

    Figure Lengend Snippet: OTUD5 interacts with UBR5 and SPT16 through distinct regions. ( A ) PONDR software predicts the disordered regions within OTUD5 protein. Below is a schematic for the OTUD5 truncation plasmids used for interaction and other functional analyses. The summary of the assay results are shown on the right: ‘Binding’ was measured by co-IP assays as shown in ( B ), and ‘Stabilization’ was measured by western blots as shown in ( C ). ‘NLS’ is indicated on the schematics based on our analysis in . ( B ) 293T cells transfected with the indicated plasmids, then anti-FLAG IP was performed and the eluates were analyzed by western blotting ( C ). HeLa OTUD5 KO (clone#2) cells were transfected with indicated plasmids and UBR5 protein was analyzed. ( D ) Cross-species alignment on the N-terminus of OTUD5. ( E ) Purified WT or ΔUIM GST-OTUD5 recombinant proteins was mixed with 293T cell lysate, pulled down with glutathione beads, then the eluates were analyzed by western blots. ( F ) Ptuner263 cells were transfected with indicated OTUD5 truncates (EV = empty vector) then the Fok1 nucleases were induced by Shield1 and 4-OHT. Cells were fixed then stained with anti-FLAG antibody. The experiments were done in triplicates and on the right is the statistical information ( N = 30 each). ( G ) HeLa cells were transfected with indicated pBABE-FLAG-OTUD5 plasmids (NT = non transfected, EV = empty vector) and the PLA assay with anti-UBR5 and anti-FLAG (OTUD5) was performed ( N = 30). Scale bars indicate 10 μm. (**** indicates P -value < 0.0005, N.S. = not significant).

    Article Snippet: The U2OS cells stably expressing SNAP-H2A were generated through G418 selection and clonal isolation. pDEST_Tet_CMV_FLAG-OTUD5 was a gift from Wade Harper (Addgene plasmid # 22610) ( ) and was used for generating HeLa cells stably expressing FLAG-OTUD5. pDEST10-6xHis-UBR5 plasmid was a gift from Sally Kornbluth ( ) and was used for recombinant protein production from SF9 cells.

    Techniques: Software, Functional Assay, Binding Assay, Co-Immunoprecipitation Assay, Western Blot, Transfection, Purification, Recombinant, Plasmid Preparation, Staining

    Interaction-deficient OTUD5 mutants cannot rescue the transcription repressive phenotypes. The PTuner 263 cells expressing indicated OTUD5 constructs (EV = empty vector) were transfected with siRNAs targeting the 3’UTR region of OTUD5, treated with 4-OHT and Shield-1 to stabilize Fok1, then the presence of YFP-MS2 expression at the Fok1 spots was measured. YFP-MS2 reporter was induced by tetracycline 3 hours prior to fixing. Only the cells with positive FLAG spots were counted. The assay was performed in triplicates ( N = 50). On the center is the quantification of the MS2 RFI at Fok1 nuclease using Image J ( N = 50). Below is the expression confirmation of each cell. Scale bars indicate 10 μm. (**** indicates P -value < 0.0005, ** <0.05.)

    Journal: Nucleic Acids Research

    Article Title: The OTUD5–UBR5 complex regulates FACT-mediated transcription at damaged chromatin

    doi: 10.1093/nar/gky1219

    Figure Lengend Snippet: Interaction-deficient OTUD5 mutants cannot rescue the transcription repressive phenotypes. The PTuner 263 cells expressing indicated OTUD5 constructs (EV = empty vector) were transfected with siRNAs targeting the 3’UTR region of OTUD5, treated with 4-OHT and Shield-1 to stabilize Fok1, then the presence of YFP-MS2 expression at the Fok1 spots was measured. YFP-MS2 reporter was induced by tetracycline 3 hours prior to fixing. Only the cells with positive FLAG spots were counted. The assay was performed in triplicates ( N = 50). On the center is the quantification of the MS2 RFI at Fok1 nuclease using Image J ( N = 50). Below is the expression confirmation of each cell. Scale bars indicate 10 μm. (**** indicates P -value < 0.0005, ** <0.05.)

    Article Snippet: The U2OS cells stably expressing SNAP-H2A were generated through G418 selection and clonal isolation. pDEST_Tet_CMV_FLAG-OTUD5 was a gift from Wade Harper (Addgene plasmid # 22610) ( ) and was used for generating HeLa cells stably expressing FLAG-OTUD5. pDEST10-6xHis-UBR5 plasmid was a gift from Sally Kornbluth ( ) and was used for recombinant protein production from SF9 cells.

    Techniques: Expressing, Construct, Plasmid Preparation, Transfection

    Cancer-associated OTUD5 missense mutation disrupts the FACT association. ( A ) Sequence alignment of the UIM and indication of a cancer-associated missense mutation (D537A; Cbioportal). ( B ) anti-FLAG IPs were performed from 293T cells transfected with indicated OTUD5 constructs (EV = empty vector) and the eluates were analyzed by western blotting. ( C ) The PTuner 263 cells transfected with the indicated FLAG-OTUD5 constructs were treated with siRNA targeting the 3’UTR region of OTUD5, treated with 4-OHT and Shield-1 to stabilize Fok1, then stained with FLAG antibody. YFP-MS2 reporter transcription was induced by tetracycline 3 hour prior to fixing. The intensities (RFI) of YFP-MS2 expression at the FLAG-positive Fok1 sites was measured using Image J, then plotted on the right. The assay was performed in triplicates ( N = 50). Scale bars indicate 10 μm. (**** indicates P -value < 0.0005, ** <0.05).

    Journal: Nucleic Acids Research

    Article Title: The OTUD5–UBR5 complex regulates FACT-mediated transcription at damaged chromatin

    doi: 10.1093/nar/gky1219

    Figure Lengend Snippet: Cancer-associated OTUD5 missense mutation disrupts the FACT association. ( A ) Sequence alignment of the UIM and indication of a cancer-associated missense mutation (D537A; Cbioportal). ( B ) anti-FLAG IPs were performed from 293T cells transfected with indicated OTUD5 constructs (EV = empty vector) and the eluates were analyzed by western blotting. ( C ) The PTuner 263 cells transfected with the indicated FLAG-OTUD5 constructs were treated with siRNA targeting the 3’UTR region of OTUD5, treated with 4-OHT and Shield-1 to stabilize Fok1, then stained with FLAG antibody. YFP-MS2 reporter transcription was induced by tetracycline 3 hour prior to fixing. The intensities (RFI) of YFP-MS2 expression at the FLAG-positive Fok1 sites was measured using Image J, then plotted on the right. The assay was performed in triplicates ( N = 50). Scale bars indicate 10 μm. (**** indicates P -value < 0.0005, ** <0.05).

    Article Snippet: The U2OS cells stably expressing SNAP-H2A were generated through G418 selection and clonal isolation. pDEST_Tet_CMV_FLAG-OTUD5 was a gift from Wade Harper (Addgene plasmid # 22610) ( ) and was used for generating HeLa cells stably expressing FLAG-OTUD5. pDEST10-6xHis-UBR5 plasmid was a gift from Sally Kornbluth ( ) and was used for recombinant protein production from SF9 cells.

    Techniques: Mutagenesis, Sequencing, Transfection, Construct, Plasmid Preparation, Western Blot, Staining, Expressing

    OTUD5 depletion causes genome instability. U2OS ( A ) and HeLa ( B ) cells were transfected with indicated siRNAs, then 48 hours later treated with indicated concentrations of Bleomycin. Cells were incubated for 10 additional days, fixed, then stained using crystal violet. The staining intensities measured by colorimetric analysis as described in ‘Materials and Methods’ section. Assays were done in triplicates. ( C ) U2OS cells were transfected with siRNAs, then treated with Bleomycin (5 µM) for 16 hours before fixed for flow cytometer analysis. On the right is the quantification of the subG1 populations. The assays were done in triplicates. ( D ) HeLa WT or OTUD5 KO cells were fixed and pRPA32 foci were counted using Image J ( N = 50). (*** indicates P -value < 0.005).

    Journal: Nucleic Acids Research

    Article Title: The OTUD5–UBR5 complex regulates FACT-mediated transcription at damaged chromatin

    doi: 10.1093/nar/gky1219

    Figure Lengend Snippet: OTUD5 depletion causes genome instability. U2OS ( A ) and HeLa ( B ) cells were transfected with indicated siRNAs, then 48 hours later treated with indicated concentrations of Bleomycin. Cells were incubated for 10 additional days, fixed, then stained using crystal violet. The staining intensities measured by colorimetric analysis as described in ‘Materials and Methods’ section. Assays were done in triplicates. ( C ) U2OS cells were transfected with siRNAs, then treated with Bleomycin (5 µM) for 16 hours before fixed for flow cytometer analysis. On the right is the quantification of the subG1 populations. The assays were done in triplicates. ( D ) HeLa WT or OTUD5 KO cells were fixed and pRPA32 foci were counted using Image J ( N = 50). (*** indicates P -value < 0.005).

    Article Snippet: The U2OS cells stably expressing SNAP-H2A were generated through G418 selection and clonal isolation. pDEST_Tet_CMV_FLAG-OTUD5 was a gift from Wade Harper (Addgene plasmid # 22610) ( ) and was used for generating HeLa cells stably expressing FLAG-OTUD5. pDEST10-6xHis-UBR5 plasmid was a gift from Sally Kornbluth ( ) and was used for recombinant protein production from SF9 cells.

    Techniques: Transfection, Incubation, Staining, Flow Cytometry