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  • 99
    Millipore mgcl2
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    Cell Signaling Technology Inc anti parp1
    Smurf2 mediates <t>PARP1</t> degradation through proteasome pathway. A, Overexpression of Flag‐Smurf2 and Flag‐Smurf2 (C716G) in HUVEC stably knocking down of Smurf2, Flag‐Smurf2 (C716G) lost the negative regulation of PARP1 by Western blot. β‐tubulin was used as a loading control. B, Transfection of 293T cells with Flag‐Smurf2 and Flag‐Smurf2 (C716G) in 293T cells together with Myc‐PARP1 for 48 h, anti‐Flag antibody and Protein A/G immunoprecipitation magnetic beads followed by anti‐Myc Western blot, the interaction between Flag‐Smurf2 (C716G) and Myc‐PARP1 is weakened. C, Transfection of 293T cells with Flag‐Smurf2 and cells were treated with CHX at 100 μmol/L or DMSO for the indicated times. The half‐life of PARP1 was measured by Western blot. β‐tubulin was used as a loading control, and each point is represented as the mean ± SD of triplicate experiments. D, Knockdown Smurf2 with control siRNA and Smurf2 siRNA in 293T cells were treated with CHX at 100 μmol/L or DMSO for the indicated times. The half‐life of PARP1 was measured by Western blot. β‐tubulin was used as a loading control, and each point is represented as the mean ± SD of triplicate experiments. E, Transfection of Flag‐Smurf2 in 293T cells together with Myc‐PARP1, the cells were treated with CHX at 100 μmol/L or DMSO for the indicated times. The half‐life of Myc‐PARP1 was measured by Western blot. β‐tubulin was used as a loading control, and each point is represented as the mean ± SD of triplicate experiments. F, Transfection of Flag‐Smurf2 in 293T cells and cells were treated with MG132 at 25 μmol/L or DMSO for the indicated times. The accumulation of PARP1 was measured by Western blot. β‐tubulin was used as a loading control, and each point is represented as the mean ± SD of triplicate experiments. G, Knockdown Smurf2 with control siRNA and Smurf2 siRNA in 293T cells were treated with MG132 at 25 μmol/L or DMSO for the indicated times. The accumulation of PARP1 was measured by Western blot. β‐tubulin was used as a loading control and each point is represented as the mean ± SD of triplicate experiments
    Anti Parp1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 307 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Transcription output analysis of the tricho-rhino-phalangeal syndrome 1 (TRPSI)-ubiquitin-specific protease 4 (USP4)-histone deacetylase 2 <t>(HDAC2)</t> regulatory axis. a Analysis and comparison of transcriptomes of MCF7 upon silencing TRPS1 or HDAC2 using RNA sequencing. b Validation of the selected TRPS1 target genes using RT-qPCR. c Validation the selected HDAC2 target genes using RT-qPCR. d Expression levels of genes up-regulated upon silencing of TRPS1 were restored with overexpression of HDAC2. e Selected genes upon silencing of TRPS1 or HDAC2 show consistent up-regulation by RT-qPCR upon silencing of USP4 in MCF7. f Expression levels of genes up-regulated upon silencing of USP4 were restored with additional overexpression of HDAC2. g , h Chromatin immunoprecipitation (ChIP)-qPCR using H4K16AC antibodies on selected target genes upon silencing of TRPS1 or USP4 in MCF7 cells. The t test was used for statistical quantifications: * p
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    Biotools B & M Labs biotools dna polymerase
    Transcription output analysis of the tricho-rhino-phalangeal syndrome 1 (TRPSI)-ubiquitin-specific protease 4 (USP4)-histone deacetylase 2 <t>(HDAC2)</t> regulatory axis. a Analysis and comparison of transcriptomes of MCF7 upon silencing TRPS1 or HDAC2 using RNA sequencing. b Validation of the selected TRPS1 target genes using RT-qPCR. c Validation the selected HDAC2 target genes using RT-qPCR. d Expression levels of genes up-regulated upon silencing of TRPS1 were restored with overexpression of HDAC2. e Selected genes upon silencing of TRPS1 or HDAC2 show consistent up-regulation by RT-qPCR upon silencing of USP4 in MCF7. f Expression levels of genes up-regulated upon silencing of USP4 were restored with additional overexpression of HDAC2. g , h Chromatin immunoprecipitation (ChIP)-qPCR using H4K16AC antibodies on selected target genes upon silencing of TRPS1 or USP4 in MCF7 cells. The t test was used for statistical quantifications: * p
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    Transcription output analysis of the tricho-rhino-phalangeal syndrome 1 (TRPSI)-ubiquitin-specific protease 4 (USP4)-histone deacetylase 2 <t>(HDAC2)</t> regulatory axis. a Analysis and comparison of transcriptomes of MCF7 upon silencing TRPS1 or HDAC2 using RNA sequencing. b Validation of the selected TRPS1 target genes using RT-qPCR. c Validation the selected HDAC2 target genes using RT-qPCR. d Expression levels of genes up-regulated upon silencing of TRPS1 were restored with overexpression of HDAC2. e Selected genes upon silencing of TRPS1 or HDAC2 show consistent up-regulation by RT-qPCR upon silencing of USP4 in MCF7. f Expression levels of genes up-regulated upon silencing of USP4 were restored with additional overexpression of HDAC2. g , h Chromatin immunoprecipitation (ChIP)-qPCR using H4K16AC antibodies on selected target genes upon silencing of TRPS1 or USP4 in MCF7 cells. The t test was used for statistical quantifications: * p
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    Bio-Rad dna engine thermal cycler
    Transcription output analysis of the tricho-rhino-phalangeal syndrome 1 (TRPSI)-ubiquitin-specific protease 4 (USP4)-histone deacetylase 2 <t>(HDAC2)</t> regulatory axis. a Analysis and comparison of transcriptomes of MCF7 upon silencing TRPS1 or HDAC2 using RNA sequencing. b Validation of the selected TRPS1 target genes using RT-qPCR. c Validation the selected HDAC2 target genes using RT-qPCR. d Expression levels of genes up-regulated upon silencing of TRPS1 were restored with overexpression of HDAC2. e Selected genes upon silencing of TRPS1 or HDAC2 show consistent up-regulation by RT-qPCR upon silencing of USP4 in MCF7. f Expression levels of genes up-regulated upon silencing of USP4 were restored with additional overexpression of HDAC2. g , h Chromatin immunoprecipitation (ChIP)-qPCR using H4K16AC antibodies on selected target genes upon silencing of TRPS1 or USP4 in MCF7 cells. The t test was used for statistical quantifications: * p
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    Transcription output analysis of the tricho-rhino-phalangeal syndrome 1 (TRPSI)-ubiquitin-specific protease 4 (USP4)-histone deacetylase 2 <t>(HDAC2)</t> regulatory axis. a Analysis and comparison of transcriptomes of MCF7 upon silencing TRPS1 or HDAC2 using RNA sequencing. b Validation of the selected TRPS1 target genes using RT-qPCR. c Validation the selected HDAC2 target genes using RT-qPCR. d Expression levels of genes up-regulated upon silencing of TRPS1 were restored with overexpression of HDAC2. e Selected genes upon silencing of TRPS1 or HDAC2 show consistent up-regulation by RT-qPCR upon silencing of USP4 in MCF7. f Expression levels of genes up-regulated upon silencing of USP4 were restored with additional overexpression of HDAC2. g , h Chromatin immunoprecipitation (ChIP)-qPCR using H4K16AC antibodies on selected target genes upon silencing of TRPS1 or USP4 in MCF7 cells. The t test was used for statistical quantifications: * p
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    Transcription output analysis of the tricho-rhino-phalangeal syndrome 1 (TRPSI)-ubiquitin-specific protease 4 (USP4)-histone deacetylase 2 <t>(HDAC2)</t> regulatory axis. a Analysis and comparison of transcriptomes of MCF7 upon silencing TRPS1 or HDAC2 using RNA sequencing. b Validation of the selected TRPS1 target genes using RT-qPCR. c Validation the selected HDAC2 target genes using RT-qPCR. d Expression levels of genes up-regulated upon silencing of TRPS1 were restored with overexpression of HDAC2. e Selected genes upon silencing of TRPS1 or HDAC2 show consistent up-regulation by RT-qPCR upon silencing of USP4 in MCF7. f Expression levels of genes up-regulated upon silencing of USP4 were restored with additional overexpression of HDAC2. g , h Chromatin immunoprecipitation (ChIP)-qPCR using H4K16AC antibodies on selected target genes upon silencing of TRPS1 or USP4 in MCF7 cells. The t test was used for statistical quantifications: * p
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    Transcription output analysis of the tricho-rhino-phalangeal syndrome 1 (TRPSI)-ubiquitin-specific protease 4 (USP4)-histone deacetylase 2 <t>(HDAC2)</t> regulatory axis. a Analysis and comparison of transcriptomes of MCF7 upon silencing TRPS1 or HDAC2 using RNA sequencing. b Validation of the selected TRPS1 target genes using RT-qPCR. c Validation the selected HDAC2 target genes using RT-qPCR. d Expression levels of genes up-regulated upon silencing of TRPS1 were restored with overexpression of HDAC2. e Selected genes upon silencing of TRPS1 or HDAC2 show consistent up-regulation by RT-qPCR upon silencing of USP4 in MCF7. f Expression levels of genes up-regulated upon silencing of USP4 were restored with additional overexpression of HDAC2. g , h Chromatin immunoprecipitation (ChIP)-qPCR using H4K16AC antibodies on selected target genes upon silencing of TRPS1 or USP4 in MCF7 cells. The t test was used for statistical quantifications: * p
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    Transcription output analysis of the tricho-rhino-phalangeal syndrome 1 (TRPSI)-ubiquitin-specific protease 4 (USP4)-histone deacetylase 2 <t>(HDAC2)</t> regulatory axis. a Analysis and comparison of transcriptomes of MCF7 upon silencing TRPS1 or HDAC2 using RNA sequencing. b Validation of the selected TRPS1 target genes using RT-qPCR. c Validation the selected HDAC2 target genes using RT-qPCR. d Expression levels of genes up-regulated upon silencing of TRPS1 were restored with overexpression of HDAC2. e Selected genes upon silencing of TRPS1 or HDAC2 show consistent up-regulation by RT-qPCR upon silencing of USP4 in MCF7. f Expression levels of genes up-regulated upon silencing of USP4 were restored with additional overexpression of HDAC2. g , h Chromatin immunoprecipitation (ChIP)-qPCR using H4K16AC antibodies on selected target genes upon silencing of TRPS1 or USP4 in MCF7 cells. The t test was used for statistical quantifications: * p
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    Transcription output analysis of the tricho-rhino-phalangeal syndrome 1 (TRPSI)-ubiquitin-specific protease 4 (USP4)-histone deacetylase 2 <t>(HDAC2)</t> regulatory axis. a Analysis and comparison of transcriptomes of MCF7 upon silencing TRPS1 or HDAC2 using RNA sequencing. b Validation of the selected TRPS1 target genes using RT-qPCR. c Validation the selected HDAC2 target genes using RT-qPCR. d Expression levels of genes up-regulated upon silencing of TRPS1 were restored with overexpression of HDAC2. e Selected genes upon silencing of TRPS1 or HDAC2 show consistent up-regulation by RT-qPCR upon silencing of USP4 in MCF7. f Expression levels of genes up-regulated upon silencing of USP4 were restored with additional overexpression of HDAC2. g , h Chromatin immunoprecipitation (ChIP)-qPCR using H4K16AC antibodies on selected target genes upon silencing of TRPS1 or USP4 in MCF7 cells. The t test was used for statistical quantifications: * p
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    Transcription output analysis of the tricho-rhino-phalangeal syndrome 1 (TRPSI)-ubiquitin-specific protease 4 (USP4)-histone deacetylase 2 <t>(HDAC2)</t> regulatory axis. a Analysis and comparison of transcriptomes of MCF7 upon silencing TRPS1 or HDAC2 using RNA sequencing. b Validation of the selected TRPS1 target genes using RT-qPCR. c Validation the selected HDAC2 target genes using RT-qPCR. d Expression levels of genes up-regulated upon silencing of TRPS1 were restored with overexpression of HDAC2. e Selected genes upon silencing of TRPS1 or HDAC2 show consistent up-regulation by RT-qPCR upon silencing of USP4 in MCF7. f Expression levels of genes up-regulated upon silencing of USP4 were restored with additional overexpression of HDAC2. g , h Chromatin immunoprecipitation (ChIP)-qPCR using H4K16AC antibodies on selected target genes upon silencing of TRPS1 or USP4 in MCF7 cells. The t test was used for statistical quantifications: * p
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    Image Search Results


    Smurf2 mediates PARP1 degradation through proteasome pathway. A, Overexpression of Flag‐Smurf2 and Flag‐Smurf2 (C716G) in HUVEC stably knocking down of Smurf2, Flag‐Smurf2 (C716G) lost the negative regulation of PARP1 by Western blot. β‐tubulin was used as a loading control. B, Transfection of 293T cells with Flag‐Smurf2 and Flag‐Smurf2 (C716G) in 293T cells together with Myc‐PARP1 for 48 h, anti‐Flag antibody and Protein A/G immunoprecipitation magnetic beads followed by anti‐Myc Western blot, the interaction between Flag‐Smurf2 (C716G) and Myc‐PARP1 is weakened. C, Transfection of 293T cells with Flag‐Smurf2 and cells were treated with CHX at 100 μmol/L or DMSO for the indicated times. The half‐life of PARP1 was measured by Western blot. β‐tubulin was used as a loading control, and each point is represented as the mean ± SD of triplicate experiments. D, Knockdown Smurf2 with control siRNA and Smurf2 siRNA in 293T cells were treated with CHX at 100 μmol/L or DMSO for the indicated times. The half‐life of PARP1 was measured by Western blot. β‐tubulin was used as a loading control, and each point is represented as the mean ± SD of triplicate experiments. E, Transfection of Flag‐Smurf2 in 293T cells together with Myc‐PARP1, the cells were treated with CHX at 100 μmol/L or DMSO for the indicated times. The half‐life of Myc‐PARP1 was measured by Western blot. β‐tubulin was used as a loading control, and each point is represented as the mean ± SD of triplicate experiments. F, Transfection of Flag‐Smurf2 in 293T cells and cells were treated with MG132 at 25 μmol/L or DMSO for the indicated times. The accumulation of PARP1 was measured by Western blot. β‐tubulin was used as a loading control, and each point is represented as the mean ± SD of triplicate experiments. G, Knockdown Smurf2 with control siRNA and Smurf2 siRNA in 293T cells were treated with MG132 at 25 μmol/L or DMSO for the indicated times. The accumulation of PARP1 was measured by Western blot. β‐tubulin was used as a loading control and each point is represented as the mean ± SD of triplicate experiments

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: The E3 ubiquitin ligase Smurf2 regulates PARP1 stability to alleviate oxidative stress‐induced injury in human umbilical vein endothelial cells, et al. The E3 ubiquitin ligase Smurf2 regulates PARP1 stability to alleviate oxidative stress‐induced injury in human umbilical vein endothelial cells

    doi: 10.1111/jcmm.15121

    Figure Lengend Snippet: Smurf2 mediates PARP1 degradation through proteasome pathway. A, Overexpression of Flag‐Smurf2 and Flag‐Smurf2 (C716G) in HUVEC stably knocking down of Smurf2, Flag‐Smurf2 (C716G) lost the negative regulation of PARP1 by Western blot. β‐tubulin was used as a loading control. B, Transfection of 293T cells with Flag‐Smurf2 and Flag‐Smurf2 (C716G) in 293T cells together with Myc‐PARP1 for 48 h, anti‐Flag antibody and Protein A/G immunoprecipitation magnetic beads followed by anti‐Myc Western blot, the interaction between Flag‐Smurf2 (C716G) and Myc‐PARP1 is weakened. C, Transfection of 293T cells with Flag‐Smurf2 and cells were treated with CHX at 100 μmol/L or DMSO for the indicated times. The half‐life of PARP1 was measured by Western blot. β‐tubulin was used as a loading control, and each point is represented as the mean ± SD of triplicate experiments. D, Knockdown Smurf2 with control siRNA and Smurf2 siRNA in 293T cells were treated with CHX at 100 μmol/L or DMSO for the indicated times. The half‐life of PARP1 was measured by Western blot. β‐tubulin was used as a loading control, and each point is represented as the mean ± SD of triplicate experiments. E, Transfection of Flag‐Smurf2 in 293T cells together with Myc‐PARP1, the cells were treated with CHX at 100 μmol/L or DMSO for the indicated times. The half‐life of Myc‐PARP1 was measured by Western blot. β‐tubulin was used as a loading control, and each point is represented as the mean ± SD of triplicate experiments. F, Transfection of Flag‐Smurf2 in 293T cells and cells were treated with MG132 at 25 μmol/L or DMSO for the indicated times. The accumulation of PARP1 was measured by Western blot. β‐tubulin was used as a loading control, and each point is represented as the mean ± SD of triplicate experiments. G, Knockdown Smurf2 with control siRNA and Smurf2 siRNA in 293T cells were treated with MG132 at 25 μmol/L or DMSO for the indicated times. The accumulation of PARP1 was measured by Western blot. β‐tubulin was used as a loading control and each point is represented as the mean ± SD of triplicate experiments

    Article Snippet: 2.3 Antibodies and reagents The following antibodies were used: anti‐caspase3 (D3R6Y, mAb #14220, Cell Signaling Technology, WB: 1:1000), anti‐Smurf2 (D8B8, mAb #12024, Cell Signaling Technology, WB: 1:1000, IF: 1:50), anti‐PARP1 (46D11, mAb #9532, Cell Signaling Technology, WB: 1:1000, IF: 1:200), anti‐MY (9B11, mAb #2276, Cell Signaling Technology, WB: 1:1000), anti‐Flag(9A3, mAb #8146, Cell Signaling Technology, WB: 1:1000, IF: 1:50), anti‐HA (6E2, mAb #2367, Cell Signaling Technology, WB: 1:1000) and anti‐β‐tubulin (10094‐1‐AP, Proteintech, WB: 1:1000).

    Techniques: Over Expression, Stable Transfection, Western Blot, Transfection, Immunoprecipitation, Magnetic Beads

    Smurf2 degrade PARP1 by polyubiquitination. A, Transfection of Flag‐Smurf2 in 293T cells together with Myc‐PARP1, the cells were treated with MG132 at 25 μmol/L or DMSO for 8 h. Anti‐Myc antibody and Protein A/G immunoprecipitation magnetic beads followed by anti‐Flag by Western blot. B, Transfection of Flag‐Smurf2 in 293T cells and the cells were treated with MG132 at 25 μmol/L or DMSO for 8 h, and Heclin 25 μmol/L, 2 h. The accumulation of PARP1 was measured by Western blot. β‐tubulin was used as a loading control. C, Overexpression of Flag‐Smurf2 in 293T cells together with Myc‐PARP1 and HA‐UB, anti‐Myc antibody and Protein A/G immunoprecipitation magnetic beads followed by anti‐HA Western blot a to verify the ubiquitination of Myc‐PARP1. D, Transfection of Flag‐Smurf2, Myc‐PARP1 and HA‐UB in HUVEC stably knocking down of Smurf2, anti‐Myc antibody and Protein A/G immunoprecipitation magnetic beads followed by anti‐HA Western blot to verify the ubiquitination of Myc‐PARP1. E, Transfection of Flag‐Smurf2, Myc‐PARP1 and HA‐UB in 293T cells, and the cells were treated with Heclin 25 μmol/L, 2 h, anti‐Myc antibody and Protein A/G immunoprecipitation magnetic beads followed by anti‐Flag by Western blot

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: The E3 ubiquitin ligase Smurf2 regulates PARP1 stability to alleviate oxidative stress‐induced injury in human umbilical vein endothelial cells, et al. The E3 ubiquitin ligase Smurf2 regulates PARP1 stability to alleviate oxidative stress‐induced injury in human umbilical vein endothelial cells

    doi: 10.1111/jcmm.15121

    Figure Lengend Snippet: Smurf2 degrade PARP1 by polyubiquitination. A, Transfection of Flag‐Smurf2 in 293T cells together with Myc‐PARP1, the cells were treated with MG132 at 25 μmol/L or DMSO for 8 h. Anti‐Myc antibody and Protein A/G immunoprecipitation magnetic beads followed by anti‐Flag by Western blot. B, Transfection of Flag‐Smurf2 in 293T cells and the cells were treated with MG132 at 25 μmol/L or DMSO for 8 h, and Heclin 25 μmol/L, 2 h. The accumulation of PARP1 was measured by Western blot. β‐tubulin was used as a loading control. C, Overexpression of Flag‐Smurf2 in 293T cells together with Myc‐PARP1 and HA‐UB, anti‐Myc antibody and Protein A/G immunoprecipitation magnetic beads followed by anti‐HA Western blot a to verify the ubiquitination of Myc‐PARP1. D, Transfection of Flag‐Smurf2, Myc‐PARP1 and HA‐UB in HUVEC stably knocking down of Smurf2, anti‐Myc antibody and Protein A/G immunoprecipitation magnetic beads followed by anti‐HA Western blot to verify the ubiquitination of Myc‐PARP1. E, Transfection of Flag‐Smurf2, Myc‐PARP1 and HA‐UB in 293T cells, and the cells were treated with Heclin 25 μmol/L, 2 h, anti‐Myc antibody and Protein A/G immunoprecipitation magnetic beads followed by anti‐Flag by Western blot

    Article Snippet: 2.3 Antibodies and reagents The following antibodies were used: anti‐caspase3 (D3R6Y, mAb #14220, Cell Signaling Technology, WB: 1:1000), anti‐Smurf2 (D8B8, mAb #12024, Cell Signaling Technology, WB: 1:1000, IF: 1:50), anti‐PARP1 (46D11, mAb #9532, Cell Signaling Technology, WB: 1:1000, IF: 1:200), anti‐MY (9B11, mAb #2276, Cell Signaling Technology, WB: 1:1000), anti‐Flag(9A3, mAb #8146, Cell Signaling Technology, WB: 1:1000, IF: 1:50), anti‐HA (6E2, mAb #2367, Cell Signaling Technology, WB: 1:1000) and anti‐β‐tubulin (10094‐1‐AP, Proteintech, WB: 1:1000).

    Techniques: Transfection, Immunoprecipitation, Magnetic Beads, Western Blot, Over Expression, Stable Transfection

    Smurf2 can affect the stability of PARP1. A, Transfection of 293T cells with Flag‐Smurf2 for 48 h, overexpression of Smurf2 can reduce the expression of endogenous PARP1 by Western blot. β‐tubulin was used as a loading control. *** P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: The E3 ubiquitin ligase Smurf2 regulates PARP1 stability to alleviate oxidative stress‐induced injury in human umbilical vein endothelial cells, et al. The E3 ubiquitin ligase Smurf2 regulates PARP1 stability to alleviate oxidative stress‐induced injury in human umbilical vein endothelial cells

    doi: 10.1111/jcmm.15121

    Figure Lengend Snippet: Smurf2 can affect the stability of PARP1. A, Transfection of 293T cells with Flag‐Smurf2 for 48 h, overexpression of Smurf2 can reduce the expression of endogenous PARP1 by Western blot. β‐tubulin was used as a loading control. *** P

    Article Snippet: 2.3 Antibodies and reagents The following antibodies were used: anti‐caspase3 (D3R6Y, mAb #14220, Cell Signaling Technology, WB: 1:1000), anti‐Smurf2 (D8B8, mAb #12024, Cell Signaling Technology, WB: 1:1000, IF: 1:50), anti‐PARP1 (46D11, mAb #9532, Cell Signaling Technology, WB: 1:1000, IF: 1:200), anti‐MY (9B11, mAb #2276, Cell Signaling Technology, WB: 1:1000), anti‐Flag(9A3, mAb #8146, Cell Signaling Technology, WB: 1:1000, IF: 1:50), anti‐HA (6E2, mAb #2367, Cell Signaling Technology, WB: 1:1000) and anti‐β‐tubulin (10094‐1‐AP, Proteintech, WB: 1:1000).

    Techniques: Transfection, Over Expression, Expressing, Western Blot

    H 2 O 2 increased the expression of Smurf2 and apoptosis‐related proteins and decreases the viability of HUVEC. A, HUVEC was treated with H 2 O 2 at the concentrations of 0, 250, 500 and 750 μmol/L for 12 h, and cell viability was determined by CCK8 assay. B, HUVEC was treated with H 2 O 2 at the concentrations of 0, 250, 500 and 750 μmol/L for 12 h, and the expression of Smurf2, cleaved PARP1 and cleaved caspase3 was analysed by Western blot. β‐ tubulin was used as a loading control. *** P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: The E3 ubiquitin ligase Smurf2 regulates PARP1 stability to alleviate oxidative stress‐induced injury in human umbilical vein endothelial cells, et al. The E3 ubiquitin ligase Smurf2 regulates PARP1 stability to alleviate oxidative stress‐induced injury in human umbilical vein endothelial cells

    doi: 10.1111/jcmm.15121

    Figure Lengend Snippet: H 2 O 2 increased the expression of Smurf2 and apoptosis‐related proteins and decreases the viability of HUVEC. A, HUVEC was treated with H 2 O 2 at the concentrations of 0, 250, 500 and 750 μmol/L for 12 h, and cell viability was determined by CCK8 assay. B, HUVEC was treated with H 2 O 2 at the concentrations of 0, 250, 500 and 750 μmol/L for 12 h, and the expression of Smurf2, cleaved PARP1 and cleaved caspase3 was analysed by Western blot. β‐ tubulin was used as a loading control. *** P

    Article Snippet: 2.3 Antibodies and reagents The following antibodies were used: anti‐caspase3 (D3R6Y, mAb #14220, Cell Signaling Technology, WB: 1:1000), anti‐Smurf2 (D8B8, mAb #12024, Cell Signaling Technology, WB: 1:1000, IF: 1:50), anti‐PARP1 (46D11, mAb #9532, Cell Signaling Technology, WB: 1:1000, IF: 1:200), anti‐MY (9B11, mAb #2276, Cell Signaling Technology, WB: 1:1000), anti‐Flag(9A3, mAb #8146, Cell Signaling Technology, WB: 1:1000, IF: 1:50), anti‐HA (6E2, mAb #2367, Cell Signaling Technology, WB: 1:1000) and anti‐β‐tubulin (10094‐1‐AP, Proteintech, WB: 1:1000).

    Techniques: Expressing, CCK-8 Assay, Western Blot

    The interaction between PARP1 and Smurf2. A and B, Coimmunoprecipitation (co‐IP) and Western blotting (IP‐western) using anti‐Smurf2 or anti‐PARP1 antibody or negative control IgG and Protein A/G immunoprecipitation magnetic beads followed by anti‐PARP1 or anti‐Smurf2 Western blot were used to verify endogenous interaction between Smurf2 and PARP1. C, Transfection of 293T cells with Flag‐Smurf2 together with Myc‐PARP1 for 48 h, Anti‐Flag antibody and Protein A/G immunoprecipitation magnetic beads followed by anti‐Myc Western blot to verify the exogenous interaction between Flag‐Smurf2 and Myc‐PARP1. D and E, The endogenous interaction between Smurf2 and PARP1 was enhanced by treatment of 500 μmol/L H 2 O 2 for 12 h in HUVEC. F and G, Indicated truncates of PARP1 and Smurf2 were constructed according to their functional domains. Transfection of 293T with the indicated truncates of Myc‐PARP1 or Flag‐Smurf2. Coimmunoprecipitation (co‐IP) and Western blotting (IP‐western) using anti‐Myc or anti‐Flag antibody or negative control IgG and Protein A/G immunoprecipitation magnetic beads followed by anti‐Smurf2 or anti‐PARP1 Western blot were used to verify the interaction between Smurf2 and PARP1

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: The E3 ubiquitin ligase Smurf2 regulates PARP1 stability to alleviate oxidative stress‐induced injury in human umbilical vein endothelial cells, et al. The E3 ubiquitin ligase Smurf2 regulates PARP1 stability to alleviate oxidative stress‐induced injury in human umbilical vein endothelial cells

    doi: 10.1111/jcmm.15121

    Figure Lengend Snippet: The interaction between PARP1 and Smurf2. A and B, Coimmunoprecipitation (co‐IP) and Western blotting (IP‐western) using anti‐Smurf2 or anti‐PARP1 antibody or negative control IgG and Protein A/G immunoprecipitation magnetic beads followed by anti‐PARP1 or anti‐Smurf2 Western blot were used to verify endogenous interaction between Smurf2 and PARP1. C, Transfection of 293T cells with Flag‐Smurf2 together with Myc‐PARP1 for 48 h, Anti‐Flag antibody and Protein A/G immunoprecipitation magnetic beads followed by anti‐Myc Western blot to verify the exogenous interaction between Flag‐Smurf2 and Myc‐PARP1. D and E, The endogenous interaction between Smurf2 and PARP1 was enhanced by treatment of 500 μmol/L H 2 O 2 for 12 h in HUVEC. F and G, Indicated truncates of PARP1 and Smurf2 were constructed according to their functional domains. Transfection of 293T with the indicated truncates of Myc‐PARP1 or Flag‐Smurf2. Coimmunoprecipitation (co‐IP) and Western blotting (IP‐western) using anti‐Myc or anti‐Flag antibody or negative control IgG and Protein A/G immunoprecipitation magnetic beads followed by anti‐Smurf2 or anti‐PARP1 Western blot were used to verify the interaction between Smurf2 and PARP1

    Article Snippet: 2.3 Antibodies and reagents The following antibodies were used: anti‐caspase3 (D3R6Y, mAb #14220, Cell Signaling Technology, WB: 1:1000), anti‐Smurf2 (D8B8, mAb #12024, Cell Signaling Technology, WB: 1:1000, IF: 1:50), anti‐PARP1 (46D11, mAb #9532, Cell Signaling Technology, WB: 1:1000, IF: 1:200), anti‐MY (9B11, mAb #2276, Cell Signaling Technology, WB: 1:1000), anti‐Flag(9A3, mAb #8146, Cell Signaling Technology, WB: 1:1000, IF: 1:50), anti‐HA (6E2, mAb #2367, Cell Signaling Technology, WB: 1:1000) and anti‐β‐tubulin (10094‐1‐AP, Proteintech, WB: 1:1000).

    Techniques: Co-Immunoprecipitation Assay, Western Blot, Negative Control, Immunoprecipitation, Magnetic Beads, Transfection, Construct, Functional Assay

    Smurf2 can alleviate HUVEC damage caused by H 2 O 2 ‐induced oxidative stress. A, Transfection of HUVEC with Flag‐Smurf2, HUVEC was treated with H 2 O 2 at the concentrations of 0, 250, 500 and 750 μmol/L for 12 h and cell viability was determined by CCK8 assay. B, Knockdown Smurf2 with control siRNA and Smurf2 siRNA target sequences in HUVEC, HUVEC was treated with H 2 O 2 at the concentrations of 0, 250, 500 and 750 μmol/L for 12 h and cell viability was determined by CCK8 assay. C, Transfection of HUVEC with Flag‐Smurf2, HUVEC was treated with H 2 O 2 at the concentrations of 500 μmol/L, 12 h and the expression of Flag, cleaved PARP1 and cleaved caspase3 was analysed by Western blot. β‐ tubulin was used as a loading control. *** P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: The E3 ubiquitin ligase Smurf2 regulates PARP1 stability to alleviate oxidative stress‐induced injury in human umbilical vein endothelial cells, et al. The E3 ubiquitin ligase Smurf2 regulates PARP1 stability to alleviate oxidative stress‐induced injury in human umbilical vein endothelial cells

    doi: 10.1111/jcmm.15121

    Figure Lengend Snippet: Smurf2 can alleviate HUVEC damage caused by H 2 O 2 ‐induced oxidative stress. A, Transfection of HUVEC with Flag‐Smurf2, HUVEC was treated with H 2 O 2 at the concentrations of 0, 250, 500 and 750 μmol/L for 12 h and cell viability was determined by CCK8 assay. B, Knockdown Smurf2 with control siRNA and Smurf2 siRNA target sequences in HUVEC, HUVEC was treated with H 2 O 2 at the concentrations of 0, 250, 500 and 750 μmol/L for 12 h and cell viability was determined by CCK8 assay. C, Transfection of HUVEC with Flag‐Smurf2, HUVEC was treated with H 2 O 2 at the concentrations of 500 μmol/L, 12 h and the expression of Flag, cleaved PARP1 and cleaved caspase3 was analysed by Western blot. β‐ tubulin was used as a loading control. *** P

    Article Snippet: 2.3 Antibodies and reagents The following antibodies were used: anti‐caspase3 (D3R6Y, mAb #14220, Cell Signaling Technology, WB: 1:1000), anti‐Smurf2 (D8B8, mAb #12024, Cell Signaling Technology, WB: 1:1000, IF: 1:50), anti‐PARP1 (46D11, mAb #9532, Cell Signaling Technology, WB: 1:1000, IF: 1:200), anti‐MY (9B11, mAb #2276, Cell Signaling Technology, WB: 1:1000), anti‐Flag(9A3, mAb #8146, Cell Signaling Technology, WB: 1:1000, IF: 1:50), anti‐HA (6E2, mAb #2367, Cell Signaling Technology, WB: 1:1000) and anti‐β‐tubulin (10094‐1‐AP, Proteintech, WB: 1:1000).

    Techniques: Transfection, CCK-8 Assay, Expressing, Western Blot

    Transcription output analysis of the tricho-rhino-phalangeal syndrome 1 (TRPSI)-ubiquitin-specific protease 4 (USP4)-histone deacetylase 2 (HDAC2) regulatory axis. a Analysis and comparison of transcriptomes of MCF7 upon silencing TRPS1 or HDAC2 using RNA sequencing. b Validation of the selected TRPS1 target genes using RT-qPCR. c Validation the selected HDAC2 target genes using RT-qPCR. d Expression levels of genes up-regulated upon silencing of TRPS1 were restored with overexpression of HDAC2. e Selected genes upon silencing of TRPS1 or HDAC2 show consistent up-regulation by RT-qPCR upon silencing of USP4 in MCF7. f Expression levels of genes up-regulated upon silencing of USP4 were restored with additional overexpression of HDAC2. g , h Chromatin immunoprecipitation (ChIP)-qPCR using H4K16AC antibodies on selected target genes upon silencing of TRPS1 or USP4 in MCF7 cells. The t test was used for statistical quantifications: * p

    Journal: Breast Cancer Research : BCR

    Article Title: Tricho-rhino-phalangeal syndrome 1 protein functions as a scaffold required for ubiquitin-specific protease 4-directed histone deacetylase 2 de-ubiquitination and tumor growth

    doi: 10.1186/s13058-018-1018-7

    Figure Lengend Snippet: Transcription output analysis of the tricho-rhino-phalangeal syndrome 1 (TRPSI)-ubiquitin-specific protease 4 (USP4)-histone deacetylase 2 (HDAC2) regulatory axis. a Analysis and comparison of transcriptomes of MCF7 upon silencing TRPS1 or HDAC2 using RNA sequencing. b Validation of the selected TRPS1 target genes using RT-qPCR. c Validation the selected HDAC2 target genes using RT-qPCR. d Expression levels of genes up-regulated upon silencing of TRPS1 were restored with overexpression of HDAC2. e Selected genes upon silencing of TRPS1 or HDAC2 show consistent up-regulation by RT-qPCR upon silencing of USP4 in MCF7. f Expression levels of genes up-regulated upon silencing of USP4 were restored with additional overexpression of HDAC2. g , h Chromatin immunoprecipitation (ChIP)-qPCR using H4K16AC antibodies on selected target genes upon silencing of TRPS1 or USP4 in MCF7 cells. The t test was used for statistical quantifications: * p

    Article Snippet: Antibodies Antibodies used in this study and their sources are as follows: anti-TRPS1 (R & D Systems#AF4838), anti-HDAC2 (Cell Signaling Technology#5113), anti-H4K16ac (Millipore#39929), anti-H4 (Millipore#04–858), anti-USP4 (Cell Signaling Technology#2651), anti-actin (proteintech#60008–1-Ig), anti-HA (Biotool#B23402), anti-Flag (Sigma#F7425), anti-Myc (Biotool#B23402), anti-His (Cell Signaling Technology#12698), and anti-Gal4 (Santa Cruz#510).

    Techniques: Histone Deacetylase Assay, RNA Sequencing Assay, Quantitative RT-PCR, Expressing, Over Expression, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    The tricho-rhino-phalangeal syndrome 1 (TRPSI)-ubiquitin-specific protease 4 (USP4)-histone deacetylase 2 (HDAC2) regulatory axis confers growth in cancer cells in vitro and in xenografted tumors in vivo. a MCF7 shows decreased cell viability upon silencing of TRPS1 and additional overexpression of HDAC2 restored cell viability. b MCF7 shows decreased cell viability upon silencing of USP4 and additional overexpression of HDAC2 restored cell viability. c Xenografted tumor growth curves (left), xenografted tumor weight (right). d Representative xenografted tumors from mouse models. e Representative western blot data of selected xenografted tumors show TRPS1 and myc-HDAC2 protein levels. f A working model of the current study. The t test was used for statistic quantifications: * p

    Journal: Breast Cancer Research : BCR

    Article Title: Tricho-rhino-phalangeal syndrome 1 protein functions as a scaffold required for ubiquitin-specific protease 4-directed histone deacetylase 2 de-ubiquitination and tumor growth

    doi: 10.1186/s13058-018-1018-7

    Figure Lengend Snippet: The tricho-rhino-phalangeal syndrome 1 (TRPSI)-ubiquitin-specific protease 4 (USP4)-histone deacetylase 2 (HDAC2) regulatory axis confers growth in cancer cells in vitro and in xenografted tumors in vivo. a MCF7 shows decreased cell viability upon silencing of TRPS1 and additional overexpression of HDAC2 restored cell viability. b MCF7 shows decreased cell viability upon silencing of USP4 and additional overexpression of HDAC2 restored cell viability. c Xenografted tumor growth curves (left), xenografted tumor weight (right). d Representative xenografted tumors from mouse models. e Representative western blot data of selected xenografted tumors show TRPS1 and myc-HDAC2 protein levels. f A working model of the current study. The t test was used for statistic quantifications: * p

    Article Snippet: Antibodies Antibodies used in this study and their sources are as follows: anti-TRPS1 (R & D Systems#AF4838), anti-HDAC2 (Cell Signaling Technology#5113), anti-H4K16ac (Millipore#39929), anti-H4 (Millipore#04–858), anti-USP4 (Cell Signaling Technology#2651), anti-actin (proteintech#60008–1-Ig), anti-HA (Biotool#B23402), anti-Flag (Sigma#F7425), anti-Myc (Biotool#B23402), anti-His (Cell Signaling Technology#12698), and anti-Gal4 (Santa Cruz#510).

    Techniques: Histone Deacetylase Assay, In Vitro, In Vivo, Over Expression, Western Blot

    Tricho-rhino-phalangeal syndrome 1 (TRPS1) transcription factor negatively regulates histone deacetylase 2 (HDAC2) protein level by stabilizing the HDAC2 protein. MCF7 ( a ), T47D ( b ), and BT474 ( c ) exhibit decreased HDAC2 protein levels upon silencing TRPS1 . MCF7 ( d ), T47D ( e ), and BT474 ( f ) exhibit insignificant alterations in HDAC2 messenger RNA levels upon silencing TRPS1 . MCF7 ( g ), T47D ( h ), and BT474 ( i ) show decreased HDAC2 protein stability upon silencing TRPS1 . The t test was used for statistical quantification: * p

    Journal: Breast Cancer Research : BCR

    Article Title: Tricho-rhino-phalangeal syndrome 1 protein functions as a scaffold required for ubiquitin-specific protease 4-directed histone deacetylase 2 de-ubiquitination and tumor growth

    doi: 10.1186/s13058-018-1018-7

    Figure Lengend Snippet: Tricho-rhino-phalangeal syndrome 1 (TRPS1) transcription factor negatively regulates histone deacetylase 2 (HDAC2) protein level by stabilizing the HDAC2 protein. MCF7 ( a ), T47D ( b ), and BT474 ( c ) exhibit decreased HDAC2 protein levels upon silencing TRPS1 . MCF7 ( d ), T47D ( e ), and BT474 ( f ) exhibit insignificant alterations in HDAC2 messenger RNA levels upon silencing TRPS1 . MCF7 ( g ), T47D ( h ), and BT474 ( i ) show decreased HDAC2 protein stability upon silencing TRPS1 . The t test was used for statistical quantification: * p

    Article Snippet: Antibodies Antibodies used in this study and their sources are as follows: anti-TRPS1 (R & D Systems#AF4838), anti-HDAC2 (Cell Signaling Technology#5113), anti-H4K16ac (Millipore#39929), anti-H4 (Millipore#04–858), anti-USP4 (Cell Signaling Technology#2651), anti-actin (proteintech#60008–1-Ig), anti-HA (Biotool#B23402), anti-Flag (Sigma#F7425), anti-Myc (Biotool#B23402), anti-His (Cell Signaling Technology#12698), and anti-Gal4 (Santa Cruz#510).

    Techniques: Histone Deacetylase Assay

    Tricho-rhino-phalangeal syndrome 1 (TRPS1) transcription factor stabilizes histone deacetylase 2 (HDAC2) through ubiquitin-dependent proteasomal degradation (UDPD). MCF7 ( a ), T47D ( b ), and BT474 ( c ) show decreased HDAC2 protein levels upon MG132 treatment in cells with silencing of TRPS1 . d Ectopic overexpression of TRPS1 reduced HDAC2 ubiquitination level in HEK293T. DMSO, dimethyl sulfoxide; siRNA, small interfering RNA

    Journal: Breast Cancer Research : BCR

    Article Title: Tricho-rhino-phalangeal syndrome 1 protein functions as a scaffold required for ubiquitin-specific protease 4-directed histone deacetylase 2 de-ubiquitination and tumor growth

    doi: 10.1186/s13058-018-1018-7

    Figure Lengend Snippet: Tricho-rhino-phalangeal syndrome 1 (TRPS1) transcription factor stabilizes histone deacetylase 2 (HDAC2) through ubiquitin-dependent proteasomal degradation (UDPD). MCF7 ( a ), T47D ( b ), and BT474 ( c ) show decreased HDAC2 protein levels upon MG132 treatment in cells with silencing of TRPS1 . d Ectopic overexpression of TRPS1 reduced HDAC2 ubiquitination level in HEK293T. DMSO, dimethyl sulfoxide; siRNA, small interfering RNA

    Article Snippet: Antibodies Antibodies used in this study and their sources are as follows: anti-TRPS1 (R & D Systems#AF4838), anti-HDAC2 (Cell Signaling Technology#5113), anti-H4K16ac (Millipore#39929), anti-H4 (Millipore#04–858), anti-USP4 (Cell Signaling Technology#2651), anti-actin (proteintech#60008–1-Ig), anti-HA (Biotool#B23402), anti-Flag (Sigma#F7425), anti-Myc (Biotool#B23402), anti-His (Cell Signaling Technology#12698), and anti-Gal4 (Santa Cruz#510).

    Techniques: Histone Deacetylase Assay, Over Expression, Small Interfering RNA

    Tricho-rhino-phalangeal syndrome 1 (TRPS1) transcription factor regulates the transcription repressive activity of histone deacetylase 2 (HDAC2). a , b MCF7 shows increased H4K16ac level upon silencing of TRPS1, and overexpression of HDAC2 restored H4K16ac level. c, d MCF7 shows increased H4K16ac level upon silencing USP4, and overexpression of HDAC2 restored H4K16ac level. e – i Effects of TRPS1 and its truncation mutants over the transcriptional repression activity of HDAC2. The t test was used for statistical analysis: * p

    Journal: Breast Cancer Research : BCR

    Article Title: Tricho-rhino-phalangeal syndrome 1 protein functions as a scaffold required for ubiquitin-specific protease 4-directed histone deacetylase 2 de-ubiquitination and tumor growth

    doi: 10.1186/s13058-018-1018-7

    Figure Lengend Snippet: Tricho-rhino-phalangeal syndrome 1 (TRPS1) transcription factor regulates the transcription repressive activity of histone deacetylase 2 (HDAC2). a , b MCF7 shows increased H4K16ac level upon silencing of TRPS1, and overexpression of HDAC2 restored H4K16ac level. c, d MCF7 shows increased H4K16ac level upon silencing USP4, and overexpression of HDAC2 restored H4K16ac level. e – i Effects of TRPS1 and its truncation mutants over the transcriptional repression activity of HDAC2. The t test was used for statistical analysis: * p

    Article Snippet: Antibodies Antibodies used in this study and their sources are as follows: anti-TRPS1 (R & D Systems#AF4838), anti-HDAC2 (Cell Signaling Technology#5113), anti-H4K16ac (Millipore#39929), anti-H4 (Millipore#04–858), anti-USP4 (Cell Signaling Technology#2651), anti-actin (proteintech#60008–1-Ig), anti-HA (Biotool#B23402), anti-Flag (Sigma#F7425), anti-Myc (Biotool#B23402), anti-His (Cell Signaling Technology#12698), and anti-Gal4 (Santa Cruz#510).

    Techniques: Activity Assay, Histone Deacetylase Assay, Over Expression

    Tricho-rhino-phalangeal syndrome 1 (TRPS1) transcription factor recruits ubiquitin-specific protease 4 (USP4) and histone deacetylase 2 (HDAC2) to form a complex in which HDAC2 is de-ubiquitinated by USP4. MCF7 ( a ) and T47D ( b ) show co-immunoprecipitation (Co-IP) of TRPS1, USP4 and HDAC2. MCF7 ( c ) and T47D ( d ) exhibit decreased HDAC2 protein levels upon silencing of USP4 . e Co-IP analysis of the interaction between TRPS1, USP4, and HDAC2 in HEK293T cells with ectopic overexpression of Flag-TRPS1, Flag-USP4, and Myc-HDAC2 using anti-Myc antibody. f Domain structures of TRPS1 and its truncation mutants. g Co-IP analysis of the interaction between truncation mutants of TRPS1 and USP4 and HDAC2 in HEK293T cells with ectopic overexpression of TRPS1 and its truncation mutants. h Co-IP analysis of the interaction between the GATA domain of TRPS1 and USP4 and HDAC2 in HEK293T cells with ectopic overexpression of TRPS1 GATA domain truncation mutant. i MCF7 cell lysates were subjected to immunoprecipitation with control IgG or anti-TRPS1 antibody. The immunoprecipitates bound to agarose beads were eluted by elution buffer and sequential immunoprecipitation was performed with the indicated antibodies. j MCF7 and k T47D show reduced interaction between USP4 and HDAC2 upon silencing TRPS1 in Co-IP assay using anti-HDAC2 antibody. l Overexpression of USP4 decreased HDAC2 ubiquitination level and additional overexpression of TRPS1 enhanced reduction of HDAC2 ubiquitination level in HEK293T cells

    Journal: Breast Cancer Research : BCR

    Article Title: Tricho-rhino-phalangeal syndrome 1 protein functions as a scaffold required for ubiquitin-specific protease 4-directed histone deacetylase 2 de-ubiquitination and tumor growth

    doi: 10.1186/s13058-018-1018-7

    Figure Lengend Snippet: Tricho-rhino-phalangeal syndrome 1 (TRPS1) transcription factor recruits ubiquitin-specific protease 4 (USP4) and histone deacetylase 2 (HDAC2) to form a complex in which HDAC2 is de-ubiquitinated by USP4. MCF7 ( a ) and T47D ( b ) show co-immunoprecipitation (Co-IP) of TRPS1, USP4 and HDAC2. MCF7 ( c ) and T47D ( d ) exhibit decreased HDAC2 protein levels upon silencing of USP4 . e Co-IP analysis of the interaction between TRPS1, USP4, and HDAC2 in HEK293T cells with ectopic overexpression of Flag-TRPS1, Flag-USP4, and Myc-HDAC2 using anti-Myc antibody. f Domain structures of TRPS1 and its truncation mutants. g Co-IP analysis of the interaction between truncation mutants of TRPS1 and USP4 and HDAC2 in HEK293T cells with ectopic overexpression of TRPS1 and its truncation mutants. h Co-IP analysis of the interaction between the GATA domain of TRPS1 and USP4 and HDAC2 in HEK293T cells with ectopic overexpression of TRPS1 GATA domain truncation mutant. i MCF7 cell lysates were subjected to immunoprecipitation with control IgG or anti-TRPS1 antibody. The immunoprecipitates bound to agarose beads were eluted by elution buffer and sequential immunoprecipitation was performed with the indicated antibodies. j MCF7 and k T47D show reduced interaction between USP4 and HDAC2 upon silencing TRPS1 in Co-IP assay using anti-HDAC2 antibody. l Overexpression of USP4 decreased HDAC2 ubiquitination level and additional overexpression of TRPS1 enhanced reduction of HDAC2 ubiquitination level in HEK293T cells

    Article Snippet: Antibodies Antibodies used in this study and their sources are as follows: anti-TRPS1 (R & D Systems#AF4838), anti-HDAC2 (Cell Signaling Technology#5113), anti-H4K16ac (Millipore#39929), anti-H4 (Millipore#04–858), anti-USP4 (Cell Signaling Technology#2651), anti-actin (proteintech#60008–1-Ig), anti-HA (Biotool#B23402), anti-Flag (Sigma#F7425), anti-Myc (Biotool#B23402), anti-His (Cell Signaling Technology#12698), and anti-Gal4 (Santa Cruz#510).

    Techniques: Histone Deacetylase Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Over Expression, Mutagenesis