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Anti Ube2n Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Fig. 5. (A) Western blotting with anti-TRAF6 Ab to visualize the nuclear translocation of TRAF6 in MCF-7 cells treated with IL-1β for 5 min. WBs for HDAC2 and β-tubulin were run on the cytoplasmic and nuclear extracts as a control. (B) TRAF6 interaction with NCoR in IL-1β-treated MCF7 cells by immunoprecipitation with anti-NCoR Ab. (C) Graphic representation of the NCoR protein showing the putative TRAF6 interaction motif, and sequence alignment of similar motifs found in human RIP2, IRAK2, and NCoR across species. (D) Interaction between TRAF6 and the identified interaction motif in HEK293T cells transfected with Myc-TRAF6 and wild type (Left) or PxExxDAxAxxA mutant (Right) GAL4-NCoR516-811 plasmids. Immunoprecipitation was performed either with anti-Myc, anti-GAL4, or control IgG, and blotted with anti-GAL4, anti-TRAF6, or anti-Myc Ab. (E) ChIP experiment showing rapid and transient recruitment of TRAF6 and <t>Ubc13</t> to the BMP7 promoter in response to IL-1β in MCF7 pretreated with E2 for 1 h. (F) Validation of siRNA efficiency in MCF7 cells. Cells are transfected with siRNA (siCtl, siTRAF6, or siUbc13) for 48 h and relative expression measured by RTqPCR, with normalization to b-actin. (G) BMP7 derepression by IL-1β, as measured by RT-PCR, requires both TRAF6 and Ubc13. MCF7 cells transfected with either control, TRAF6, or Ubc13 siRNA, were treated with E2 and/or IL-1β for 6 h. (H) Overexpression of dominant-negative TRAF6 or mutated TAB2 abrogates derepression by IL-1β in U2OS-ERα cells transiently transfected with BMP7promoter reporter and treated with E2 and vehicle (PBS) or IL-1β for 36 h. Luciferase activity was assayed and normalized to b-gal activity. (I) MEKK1’s ubiquitin-interacting motif (UIM) interacts with K63-linked, but not K48-linked, polyubiquitin chains. GST and GST-MEKK1-UIM proteins were expressed in bacteria, purified, and used for GST pulldown with recombinant K63- (Left) or K48-linked (Right) polyubiquitin chains, followed by western blotting with anti-ubiquitin or anti-GST Ab. (J) Ubc13 is required for accumulation of K63-linked polyubiquitin chains and for the recruitment of MEKK1 to the BMP7 promoter in MCF7 cells transfected with control or Ubc13 siRNA and pretreated with E2 for 1 h prior to treatment with IL-1β. ChIP was performed with anti-K63-ubiquitin or anti-MEKK1 Ab or protein A beads alone as control. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 vs. vehicle control, Student’s t test.
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Fig. 5. (A) Western blotting with anti-TRAF6 Ab to visualize the nuclear translocation of TRAF6 in MCF-7 cells treated with IL-1β for 5 min. WBs for HDAC2 and β-tubulin were run on the cytoplasmic and nuclear extracts as a control. (B) TRAF6 interaction with NCoR in IL-1β-treated MCF7 cells by immunoprecipitation with anti-NCoR Ab. (C) Graphic representation of the NCoR protein showing the putative TRAF6 interaction motif, and sequence alignment of similar motifs found in human RIP2, IRAK2, and NCoR across species. (D) Interaction between TRAF6 and the identified interaction motif in HEK293T cells transfected with Myc-TRAF6 and wild type (Left) or PxExxDAxAxxA mutant (Right) GAL4-NCoR516-811 plasmids. Immunoprecipitation was performed either with anti-Myc, anti-GAL4, or control IgG, and blotted with anti-GAL4, anti-TRAF6, or anti-Myc Ab. (E) ChIP experiment showing rapid and transient recruitment of TRAF6 and <t>Ubc13</t> to the BMP7 promoter in response to IL-1β in MCF7 pretreated with E2 for 1 h. (F) Validation of siRNA efficiency in MCF7 cells. Cells are transfected with siRNA (siCtl, siTRAF6, or siUbc13) for 48 h and relative expression measured by RTqPCR, with normalization to b-actin. (G) BMP7 derepression by IL-1β, as measured by RT-PCR, requires both TRAF6 and Ubc13. MCF7 cells transfected with either control, TRAF6, or Ubc13 siRNA, were treated with E2 and/or IL-1β for 6 h. (H) Overexpression of dominant-negative TRAF6 or mutated TAB2 abrogates derepression by IL-1β in U2OS-ERα cells transiently transfected with BMP7promoter reporter and treated with E2 and vehicle (PBS) or IL-1β for 36 h. Luciferase activity was assayed and normalized to b-gal activity. (I) MEKK1’s ubiquitin-interacting motif (UIM) interacts with K63-linked, but not K48-linked, polyubiquitin chains. GST and GST-MEKK1-UIM proteins were expressed in bacteria, purified, and used for GST pulldown with recombinant K63- (Left) or K48-linked (Right) polyubiquitin chains, followed by western blotting with anti-ubiquitin or anti-GST Ab. (J) Ubc13 is required for accumulation of K63-linked polyubiquitin chains and for the recruitment of MEKK1 to the BMP7 promoter in MCF7 cells transfected with control or Ubc13 siRNA and pretreated with E2 for 1 h prior to treatment with IL-1β. ChIP was performed with anti-K63-ubiquitin or anti-MEKK1 Ab or protein A beads alone as control. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 vs. vehicle control, Student’s t test.
Anti Ubc13 131 148 Rabbit Polyclonal Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Fig. 5. (A) Western blotting with anti-TRAF6 Ab to visualize the nuclear translocation of TRAF6 in MCF-7 cells treated with IL-1β for 5 min. WBs for HDAC2 and β-tubulin were run on the cytoplasmic and nuclear extracts as a control. (B) TRAF6 interaction with NCoR in IL-1β-treated MCF7 cells by immunoprecipitation with anti-NCoR Ab. (C) Graphic representation of the NCoR protein showing the putative TRAF6 interaction motif, and sequence alignment of similar motifs found in human RIP2, IRAK2, and NCoR across species. (D) Interaction between TRAF6 and the identified interaction motif in HEK293T cells transfected with Myc-TRAF6 and wild type (Left) or PxExxDAxAxxA mutant (Right) GAL4-NCoR516-811 plasmids. Immunoprecipitation was performed either with anti-Myc, anti-GAL4, or control IgG, and blotted with anti-GAL4, anti-TRAF6, or anti-Myc Ab. (E) ChIP experiment showing rapid and transient recruitment of TRAF6 and <t>Ubc13</t> to the BMP7 promoter in response to IL-1β in MCF7 pretreated with E2 for 1 h. (F) Validation of siRNA efficiency in MCF7 cells. Cells are transfected with siRNA (siCtl, siTRAF6, or siUbc13) for 48 h and relative expression measured by RTqPCR, with normalization to b-actin. (G) BMP7 derepression by IL-1β, as measured by RT-PCR, requires both TRAF6 and Ubc13. MCF7 cells transfected with either control, TRAF6, or Ubc13 siRNA, were treated with E2 and/or IL-1β for 6 h. (H) Overexpression of dominant-negative TRAF6 or mutated TAB2 abrogates derepression by IL-1β in U2OS-ERα cells transiently transfected with BMP7promoter reporter and treated with E2 and vehicle (PBS) or IL-1β for 36 h. Luciferase activity was assayed and normalized to b-gal activity. (I) MEKK1’s ubiquitin-interacting motif (UIM) interacts with K63-linked, but not K48-linked, polyubiquitin chains. GST and GST-MEKK1-UIM proteins were expressed in bacteria, purified, and used for GST pulldown with recombinant K63- (Left) or K48-linked (Right) polyubiquitin chains, followed by western blotting with anti-ubiquitin or anti-GST Ab. (J) Ubc13 is required for accumulation of K63-linked polyubiquitin chains and for the recruitment of MEKK1 to the BMP7 promoter in MCF7 cells transfected with control or Ubc13 siRNA and pretreated with E2 for 1 h prior to treatment with IL-1β. ChIP was performed with anti-K63-ubiquitin or anti-MEKK1 Ab or protein A beads alone as control. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 vs. vehicle control, Student’s t test.
10243 1 Ap, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Fig. 5. (A) Western blotting with anti-TRAF6 Ab to visualize the nuclear translocation of TRAF6 in MCF-7 cells treated with IL-1β for 5 min. WBs for HDAC2 and β-tubulin were run on the cytoplasmic and nuclear extracts as a control. (B) TRAF6 interaction with NCoR in IL-1β-treated MCF7 cells by immunoprecipitation with anti-NCoR Ab. (C) Graphic representation of the NCoR protein showing the putative TRAF6 interaction motif, and sequence alignment of similar motifs found in human RIP2, IRAK2, and NCoR across species. (D) Interaction between TRAF6 and the identified interaction motif in HEK293T cells transfected with Myc-TRAF6 and wild type (Left) or PxExxDAxAxxA mutant (Right) GAL4-NCoR516-811 plasmids. Immunoprecipitation was performed either with anti-Myc, anti-GAL4, or control IgG, and blotted with anti-GAL4, anti-TRAF6, or anti-Myc Ab. (E) ChIP experiment showing rapid and transient recruitment of TRAF6 and <t>Ubc13</t> to the BMP7 promoter in response to IL-1β in MCF7 pretreated with E2 for 1 h. (F) Validation of siRNA efficiency in MCF7 cells. Cells are transfected with siRNA (siCtl, siTRAF6, or siUbc13) for 48 h and relative expression measured by RTqPCR, with normalization to b-actin. (G) BMP7 derepression by IL-1β, as measured by RT-PCR, requires both TRAF6 and Ubc13. MCF7 cells transfected with either control, TRAF6, or Ubc13 siRNA, were treated with E2 and/or IL-1β for 6 h. (H) Overexpression of dominant-negative TRAF6 or mutated TAB2 abrogates derepression by IL-1β in U2OS-ERα cells transiently transfected with BMP7promoter reporter and treated with E2 and vehicle (PBS) or IL-1β for 36 h. Luciferase activity was assayed and normalized to b-gal activity. (I) MEKK1’s ubiquitin-interacting motif (UIM) interacts with K63-linked, but not K48-linked, polyubiquitin chains. GST and GST-MEKK1-UIM proteins were expressed in bacteria, purified, and used for GST pulldown with recombinant K63- (Left) or K48-linked (Right) polyubiquitin chains, followed by western blotting with anti-ubiquitin or anti-GST Ab. (J) Ubc13 is required for accumulation of K63-linked polyubiquitin chains and for the recruitment of MEKK1 to the BMP7 promoter in MCF7 cells transfected with control or Ubc13 siRNA and pretreated with E2 for 1 h prior to treatment with IL-1β. ChIP was performed with anti-K63-ubiquitin or anti-MEKK1 Ab or protein A beads alone as control. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 vs. vehicle control, Student’s t test.
Ap Proteintech, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Fig. 5. (A) Western blotting with anti-TRAF6 Ab to visualize the nuclear translocation of TRAF6 in MCF-7 cells treated with IL-1β for 5 min. WBs for HDAC2 and β-tubulin were run on the cytoplasmic and nuclear extracts as a control. (B) TRAF6 interaction with NCoR in IL-1β-treated MCF7 cells by immunoprecipitation with anti-NCoR Ab. (C) Graphic representation of the NCoR protein showing the putative TRAF6 interaction motif, and sequence alignment of similar motifs found in human RIP2, IRAK2, and NCoR across species. (D) Interaction between TRAF6 and the identified interaction motif in HEK293T cells transfected with Myc-TRAF6 and wild type (Left) or PxExxDAxAxxA mutant (Right) GAL4-NCoR516-811 plasmids. Immunoprecipitation was performed either with anti-Myc, anti-GAL4, or control IgG, and blotted with anti-GAL4, anti-TRAF6, or anti-Myc Ab. (E) ChIP experiment showing rapid and transient recruitment of TRAF6 and <t>Ubc13</t> to the BMP7 promoter in response to IL-1β in MCF7 pretreated with E2 for 1 h. (F) Validation of siRNA efficiency in MCF7 cells. Cells are transfected with siRNA (siCtl, siTRAF6, or siUbc13) for 48 h and relative expression measured by RTqPCR, with normalization to b-actin. (G) BMP7 derepression by IL-1β, as measured by RT-PCR, requires both TRAF6 and Ubc13. MCF7 cells transfected with either control, TRAF6, or Ubc13 siRNA, were treated with E2 and/or IL-1β for 6 h. (H) Overexpression of dominant-negative TRAF6 or mutated TAB2 abrogates derepression by IL-1β in U2OS-ERα cells transiently transfected with BMP7promoter reporter and treated with E2 and vehicle (PBS) or IL-1β for 36 h. Luciferase activity was assayed and normalized to b-gal activity. (I) MEKK1’s ubiquitin-interacting motif (UIM) interacts with K63-linked, but not K48-linked, polyubiquitin chains. GST and GST-MEKK1-UIM proteins were expressed in bacteria, purified, and used for GST pulldown with recombinant K63- (Left) or K48-linked (Right) polyubiquitin chains, followed by western blotting with anti-ubiquitin or anti-GST Ab. (J) Ubc13 is required for accumulation of K63-linked polyubiquitin chains and for the recruitment of MEKK1 to the BMP7 promoter in MCF7 cells transfected with control or Ubc13 siRNA and pretreated with E2 for 1 h prior to treatment with IL-1β. ChIP was performed with anti-K63-ubiquitin or anti-MEKK1 Ab or protein A beads alone as control. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 vs. vehicle control, Student’s t test.
Anti Ubc13, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Fig. 5. (A) Western blotting with anti-TRAF6 Ab to visualize the nuclear translocation of TRAF6 in MCF-7 cells treated with IL-1β for 5 min. WBs for HDAC2 and β-tubulin were run on the cytoplasmic and nuclear extracts as a control. (B) TRAF6 interaction with NCoR in IL-1β-treated MCF7 cells by immunoprecipitation with anti-NCoR Ab. (C) Graphic representation of the NCoR protein showing the putative TRAF6 interaction motif, and sequence alignment of similar motifs found in human RIP2, IRAK2, and NCoR across species. (D) Interaction between TRAF6 and the identified interaction motif in HEK293T cells transfected with Myc-TRAF6 and wild type (Left) or PxExxDAxAxxA mutant (Right) GAL4-NCoR516-811 plasmids. Immunoprecipitation was performed either with anti-Myc, anti-GAL4, or control IgG, and blotted with anti-GAL4, anti-TRAF6, or anti-Myc Ab. (E) ChIP experiment showing rapid and transient recruitment of TRAF6 and <t>Ubc13</t> to the BMP7 promoter in response to IL-1β in MCF7 pretreated with E2 for 1 h. (F) Validation of siRNA efficiency in MCF7 cells. Cells are transfected with siRNA (siCtl, siTRAF6, or siUbc13) for 48 h and relative expression measured by RTqPCR, with normalization to b-actin. (G) BMP7 derepression by IL-1β, as measured by RT-PCR, requires both TRAF6 and Ubc13. MCF7 cells transfected with either control, TRAF6, or Ubc13 siRNA, were treated with E2 and/or IL-1β for 6 h. (H) Overexpression of dominant-negative TRAF6 or mutated TAB2 abrogates derepression by IL-1β in U2OS-ERα cells transiently transfected with BMP7promoter reporter and treated with E2 and vehicle (PBS) or IL-1β for 36 h. Luciferase activity was assayed and normalized to b-gal activity. (I) MEKK1’s ubiquitin-interacting motif (UIM) interacts with K63-linked, but not K48-linked, polyubiquitin chains. GST and GST-MEKK1-UIM proteins were expressed in bacteria, purified, and used for GST pulldown with recombinant K63- (Left) or K48-linked (Right) polyubiquitin chains, followed by western blotting with anti-ubiquitin or anti-GST Ab. (J) Ubc13 is required for accumulation of K63-linked polyubiquitin chains and for the recruitment of MEKK1 to the BMP7 promoter in MCF7 cells transfected with control or Ubc13 siRNA and pretreated with E2 for 1 h prior to treatment with IL-1β. ChIP was performed with anti-K63-ubiquitin or anti-MEKK1 Ab or protein A beads alone as control. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 vs. vehicle control, Student’s t test.
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Fig. 5. (A) Western blotting with anti-TRAF6 Ab to visualize the nuclear translocation of TRAF6 in MCF-7 cells treated with IL-1β for 5 min. WBs for HDAC2 and β-tubulin were run on the cytoplasmic and nuclear extracts as a control. (B) TRAF6 interaction with NCoR in IL-1β-treated MCF7 cells by immunoprecipitation with anti-NCoR Ab. (C) Graphic representation of the NCoR protein showing the putative TRAF6 interaction motif, and sequence alignment of similar motifs found in human RIP2, IRAK2, and NCoR across species. (D) Interaction between TRAF6 and the identified interaction motif in HEK293T cells transfected with Myc-TRAF6 and wild type (Left) or PxExxDAxAxxA mutant (Right) GAL4-NCoR516-811 plasmids. Immunoprecipitation was performed either with anti-Myc, anti-GAL4, or control IgG, and blotted with anti-GAL4, anti-TRAF6, or anti-Myc Ab. (E) ChIP experiment showing rapid and transient recruitment of TRAF6 and Ubc13 to the BMP7 promoter in response to IL-1β in MCF7 pretreated with E2 for 1 h. (F) Validation of siRNA efficiency in MCF7 cells. Cells are transfected with siRNA (siCtl, siTRAF6, or siUbc13) for 48 h and relative expression measured by RTqPCR, with normalization to b-actin. (G) BMP7 derepression by IL-1β, as measured by RT-PCR, requires both TRAF6 and Ubc13. MCF7 cells transfected with either control, TRAF6, or Ubc13 siRNA, were treated with E2 and/or IL-1β for 6 h. (H) Overexpression of dominant-negative TRAF6 or mutated TAB2 abrogates derepression by IL-1β in U2OS-ERα cells transiently transfected with BMP7promoter reporter and treated with E2 and vehicle (PBS) or IL-1β for 36 h. Luciferase activity was assayed and normalized to b-gal activity. (I) MEKK1’s ubiquitin-interacting motif (UIM) interacts with K63-linked, but not K48-linked, polyubiquitin chains. GST and GST-MEKK1-UIM proteins were expressed in bacteria, purified, and used for GST pulldown with recombinant K63- (Left) or K48-linked (Right) polyubiquitin chains, followed by western blotting with anti-ubiquitin or anti-GST Ab. (J) Ubc13 is required for accumulation of K63-linked polyubiquitin chains and for the recruitment of MEKK1 to the BMP7 promoter in MCF7 cells transfected with control or Ubc13 siRNA and pretreated with E2 for 1 h prior to treatment with IL-1β. ChIP was performed with anti-K63-ubiquitin or anti-MEKK1 Ab or protein A beads alone as control. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 vs. vehicle control, Student’s t test.

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

Article Title: Nonproteolytic ubiquitination regulates chromatin occupancy by the NCoR/SMRT/HDAC3 corepressor complex in MCF-7 breast cancer cells.

doi: 10.1073/pnas.2502805122

Figure Lengend Snippet: Fig. 5. (A) Western blotting with anti-TRAF6 Ab to visualize the nuclear translocation of TRAF6 in MCF-7 cells treated with IL-1β for 5 min. WBs for HDAC2 and β-tubulin were run on the cytoplasmic and nuclear extracts as a control. (B) TRAF6 interaction with NCoR in IL-1β-treated MCF7 cells by immunoprecipitation with anti-NCoR Ab. (C) Graphic representation of the NCoR protein showing the putative TRAF6 interaction motif, and sequence alignment of similar motifs found in human RIP2, IRAK2, and NCoR across species. (D) Interaction between TRAF6 and the identified interaction motif in HEK293T cells transfected with Myc-TRAF6 and wild type (Left) or PxExxDAxAxxA mutant (Right) GAL4-NCoR516-811 plasmids. Immunoprecipitation was performed either with anti-Myc, anti-GAL4, or control IgG, and blotted with anti-GAL4, anti-TRAF6, or anti-Myc Ab. (E) ChIP experiment showing rapid and transient recruitment of TRAF6 and Ubc13 to the BMP7 promoter in response to IL-1β in MCF7 pretreated with E2 for 1 h. (F) Validation of siRNA efficiency in MCF7 cells. Cells are transfected with siRNA (siCtl, siTRAF6, or siUbc13) for 48 h and relative expression measured by RTqPCR, with normalization to b-actin. (G) BMP7 derepression by IL-1β, as measured by RT-PCR, requires both TRAF6 and Ubc13. MCF7 cells transfected with either control, TRAF6, or Ubc13 siRNA, were treated with E2 and/or IL-1β for 6 h. (H) Overexpression of dominant-negative TRAF6 or mutated TAB2 abrogates derepression by IL-1β in U2OS-ERα cells transiently transfected with BMP7promoter reporter and treated with E2 and vehicle (PBS) or IL-1β for 36 h. Luciferase activity was assayed and normalized to b-gal activity. (I) MEKK1’s ubiquitin-interacting motif (UIM) interacts with K63-linked, but not K48-linked, polyubiquitin chains. GST and GST-MEKK1-UIM proteins were expressed in bacteria, purified, and used for GST pulldown with recombinant K63- (Left) or K48-linked (Right) polyubiquitin chains, followed by western blotting with anti-ubiquitin or anti-GST Ab. (J) Ubc13 is required for accumulation of K63-linked polyubiquitin chains and for the recruitment of MEKK1 to the BMP7 promoter in MCF7 cells transfected with control or Ubc13 siRNA and pretreated with E2 for 1 h prior to treatment with IL-1β. ChIP was performed with anti-K63-ubiquitin or anti-MEKK1 Ab or protein A beads alone as control. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 vs. vehicle control, Student’s t test.

Article Snippet: Commercial antibodies used for western blotting, immunoprecipitation, and ChIP experiments include HDAC3 rabbit polyclonal antibody #ab7030 (Abcam); anti- HDAC3(H- 99) rabbit polyclonal antibody #sc- 11417 (Santa Cruz Biotechnology); anti- ubiquitin mouse monoclonal antibody (P4D1 clone, Cell Signaling Technology); anti- β- tubulin mouse monoclonal antibody #T0198 (Sigma- Aldrich); anti- HDAC2 rabbit polyclonal antibody #ab16032 (Abcam); anti- TAB2 mouse monoclonal antibody sc- 398188, goat polyclonal antibody sc- 11850, and rabbit polyclonal sc- 20756 (Santa Cruz Biotechnology); anti- K63 ubiquitin rabbit monoclonal antibody #05- 1308 (Millipore); anti- TRAF6 rabbit polyclonal antibody sc- 7221 (Santa Cruz Biotechnology); anti- Ubc13 rabbit polyclonal antibody #10243- 1- AP (ProteinTech); anti- Ubc13 (131- 148) rabbit polyclonal antibody #PA1- 41188 (Thermo Scientific); anti- FLAG mouse monoclonal antibody, clone M2, #F3165, and anti- Flag- HRP #A8592 (Sigma- Aldrich); antiMYC(9E10) mouse monoclonal antibody #sc- 40 (Santa Cruz Biotechnology); and anti- GST(56C1) mouse monoclonal antibody #sc- 80998 (Santa Cruz Biotechnology).

Techniques: Western Blot, Translocation Assay, Control, Immunoprecipitation, Sequencing, Transfection, Mutagenesis, Biomarker Discovery, Expressing, Reverse Transcription Polymerase Chain Reaction, Over Expression, Dominant Negative Mutation, Luciferase, Activity Assay, Ubiquitin Proteomics, Bacteria, Purification, Recombinant

Fig. 5. (A) Western blotting with anti-TRAF6 Ab to visualize the nuclear translocation of TRAF6 in MCF-7 cells treated with IL-1β for 5 min. WBs for HDAC2 and β-tubulin were run on the cytoplasmic and nuclear extracts as a control. (B) TRAF6 interaction with NCoR in IL-1β-treated MCF7 cells by immunoprecipitation with anti-NCoR Ab. (C) Graphic representation of the NCoR protein showing the putative TRAF6 interaction motif, and sequence alignment of similar motifs found in human RIP2, IRAK2, and NCoR across species. (D) Interaction between TRAF6 and the identified interaction motif in HEK293T cells transfected with Myc-TRAF6 and wild type (Left) or PxExxDAxAxxA mutant (Right) GAL4-NCoR516-811 plasmids. Immunoprecipitation was performed either with anti-Myc, anti-GAL4, or control IgG, and blotted with anti-GAL4, anti-TRAF6, or anti-Myc Ab. (E) ChIP experiment showing rapid and transient recruitment of TRAF6 and Ubc13 to the BMP7 promoter in response to IL-1β in MCF7 pretreated with E2 for 1 h. (F) Validation of siRNA efficiency in MCF7 cells. Cells are transfected with siRNA (siCtl, siTRAF6, or siUbc13) for 48 h and relative expression measured by RTqPCR, with normalization to b-actin. (G) BMP7 derepression by IL-1β, as measured by RT-PCR, requires both TRAF6 and Ubc13. MCF7 cells transfected with either control, TRAF6, or Ubc13 siRNA, were treated with E2 and/or IL-1β for 6 h. (H) Overexpression of dominant-negative TRAF6 or mutated TAB2 abrogates derepression by IL-1β in U2OS-ERα cells transiently transfected with BMP7promoter reporter and treated with E2 and vehicle (PBS) or IL-1β for 36 h. Luciferase activity was assayed and normalized to b-gal activity. (I) MEKK1’s ubiquitin-interacting motif (UIM) interacts with K63-linked, but not K48-linked, polyubiquitin chains. GST and GST-MEKK1-UIM proteins were expressed in bacteria, purified, and used for GST pulldown with recombinant K63- (Left) or K48-linked (Right) polyubiquitin chains, followed by western blotting with anti-ubiquitin or anti-GST Ab. (J) Ubc13 is required for accumulation of K63-linked polyubiquitin chains and for the recruitment of MEKK1 to the BMP7 promoter in MCF7 cells transfected with control or Ubc13 siRNA and pretreated with E2 for 1 h prior to treatment with IL-1β. ChIP was performed with anti-K63-ubiquitin or anti-MEKK1 Ab or protein A beads alone as control. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 vs. vehicle control, Student’s t test.

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

Article Title: Nonproteolytic ubiquitination regulates chromatin occupancy by the NCoR/SMRT/HDAC3 corepressor complex in MCF-7 breast cancer cells.

doi: 10.1073/pnas.2502805122

Figure Lengend Snippet: Fig. 5. (A) Western blotting with anti-TRAF6 Ab to visualize the nuclear translocation of TRAF6 in MCF-7 cells treated with IL-1β for 5 min. WBs for HDAC2 and β-tubulin were run on the cytoplasmic and nuclear extracts as a control. (B) TRAF6 interaction with NCoR in IL-1β-treated MCF7 cells by immunoprecipitation with anti-NCoR Ab. (C) Graphic representation of the NCoR protein showing the putative TRAF6 interaction motif, and sequence alignment of similar motifs found in human RIP2, IRAK2, and NCoR across species. (D) Interaction between TRAF6 and the identified interaction motif in HEK293T cells transfected with Myc-TRAF6 and wild type (Left) or PxExxDAxAxxA mutant (Right) GAL4-NCoR516-811 plasmids. Immunoprecipitation was performed either with anti-Myc, anti-GAL4, or control IgG, and blotted with anti-GAL4, anti-TRAF6, or anti-Myc Ab. (E) ChIP experiment showing rapid and transient recruitment of TRAF6 and Ubc13 to the BMP7 promoter in response to IL-1β in MCF7 pretreated with E2 for 1 h. (F) Validation of siRNA efficiency in MCF7 cells. Cells are transfected with siRNA (siCtl, siTRAF6, or siUbc13) for 48 h and relative expression measured by RTqPCR, with normalization to b-actin. (G) BMP7 derepression by IL-1β, as measured by RT-PCR, requires both TRAF6 and Ubc13. MCF7 cells transfected with either control, TRAF6, or Ubc13 siRNA, were treated with E2 and/or IL-1β for 6 h. (H) Overexpression of dominant-negative TRAF6 or mutated TAB2 abrogates derepression by IL-1β in U2OS-ERα cells transiently transfected with BMP7promoter reporter and treated with E2 and vehicle (PBS) or IL-1β for 36 h. Luciferase activity was assayed and normalized to b-gal activity. (I) MEKK1’s ubiquitin-interacting motif (UIM) interacts with K63-linked, but not K48-linked, polyubiquitin chains. GST and GST-MEKK1-UIM proteins were expressed in bacteria, purified, and used for GST pulldown with recombinant K63- (Left) or K48-linked (Right) polyubiquitin chains, followed by western blotting with anti-ubiquitin or anti-GST Ab. (J) Ubc13 is required for accumulation of K63-linked polyubiquitin chains and for the recruitment of MEKK1 to the BMP7 promoter in MCF7 cells transfected with control or Ubc13 siRNA and pretreated with E2 for 1 h prior to treatment with IL-1β. ChIP was performed with anti-K63-ubiquitin or anti-MEKK1 Ab or protein A beads alone as control. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 vs. vehicle control, Student’s t test.

Article Snippet: Commercial antibodies used for western blotting, immunoprecipitation, and ChIP experiments include HDAC3 rabbit polyclonal antibody #ab7030 (Abcam); anti- HDAC3(H- 99) rabbit polyclonal antibody #sc- 11417 (Santa Cruz Biotechnology); anti- ubiquitin mouse monoclonal antibody (P4D1 clone, Cell Signaling Technology); anti- β- tubulin mouse monoclonal antibody #T0198 (Sigma- Aldrich); anti- HDAC2 rabbit polyclonal antibody #ab16032 (Abcam); anti- TAB2 mouse monoclonal antibody sc- 398188, goat polyclonal antibody sc- 11850, and rabbit polyclonal sc- 20756 (Santa Cruz Biotechnology); anti- K63 ubiquitin rabbit monoclonal antibody #05- 1308 (Millipore); anti- TRAF6 rabbit polyclonal antibody sc- 7221 (Santa Cruz Biotechnology); anti- Ubc13 rabbit polyclonal antibody #10243- 1- AP (ProteinTech); anti- Ubc13 (131- 148) rabbit polyclonal antibody #PA1- 41188 (Thermo Scientific); anti- FLAG mouse monoclonal antibody, clone M2, #F3165, and anti- Flag- HRP #A8592 (Sigma- Aldrich); antiMYC(9E10) mouse monoclonal antibody #sc- 40 (Santa Cruz Biotechnology); and anti- GST(56C1) mouse monoclonal antibody #sc- 80998 (Santa Cruz Biotechnology).

Techniques: Western Blot, Translocation Assay, Control, Immunoprecipitation, Sequencing, Transfection, Mutagenesis, Biomarker Discovery, Expressing, Reverse Transcription Polymerase Chain Reaction, Over Expression, Dominant Negative Mutation, Luciferase, Activity Assay, Ubiquitin Proteomics, Bacteria, Purification, Recombinant