Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: PCV2 infection led to translocation of HMGB1 from nuclei to cytoplasmic compartments. PK-15 cells and porcine monocytic cells (3D4/31) were infected for 36 h with PCV2 (MOI = 1) or mock infected as a control. (A) Confocal imaging of HMGB1 distribution in PCV2-infected cells immunostained with anti-HMGB1 (green) and anti-Cap (red) antibodies. Nuclei were labeled with DAPI (blue). Representative micrographic images are shown. (B) Immunoblotting of PCV2 Cap and HMGB1 in nuclear and cytoplasmic extracts from PCV2- or mock-infected PK-15 cells. Histone H3 and GAPDH were used as internal controls for nuclear and cytoplasmic fractions, respectively. (C) The intensity of protein bands was quantified densitometrically using Gel-Pro Analyzer. Ratios of nuclear or cytoplasmic HMGB1 to Histone H3 or GAPDH were quantified, respectively. (D and E) Quantification of hmgb1 mRNA by qPCR in PK-15 and 3D4/31 cells infected with PCV2 for different times using total RNA extracts from the cells. (F and G) Immunoblotting of HMGB1 and PCV2 Cap in the lysates of PK-15 and 3D4/31 cells infected with PCV2 for different times. β-Actin was used as a loading control. The data in panels A, B, F, and G are representative of three independent experiments. Bar charts in panels C, D, and E show means ± SDs from three independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Infection, Translocation Assay, Imaging, Labeling, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Overexpression of HMGB1 inhibited PCV2 replication. PK-15 cells were transfected with recombinant plasmid expressing HMGB1 (pHMGB1) or control plasmid (pFlag) for 24 h and then infected with PCV2 (MOI = 1) for 36 h. (A) Effect of HMGB1 overexpression on PCV2 Cap expression as shown by immunoblotting using protein samples from the whole-cell lysates. β-Actin was used as a loading control. The gel shown is representative of three independent experiments. (B) The ratios of band intensity of HMGB1 or PCV2 Cap to β-actin (as shown in panel A). (C) Effect of HMGB1 overexpression on PCV2 orf2 (encoding Cap) transcription measured by qPCR using total RNA extracted from the whole-cell lysates. (D) PCV2 replication in cells overexpressing HMGB1 as assessed by indirect immunofluorescence. Percentages of PCV2-infected cells were calculated as described in the legend for Fig. 2. Relative percentages of PCV2-infected cells in the HMGB1 overexpressing cells are shown with nontransfected but PCV2-infected cells set at 100%. (E) PCV2 genomic DNA copies in cells overexpressing HMGB1 quantified by qPCR using total DNA extracts from whole-cell lysates. Bar charts in panels B, C, D, and E show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01; ***, P < 0.001.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Over Expression, Transfection, Recombinant, Plasmid Preparation, Expressing, Infection, Western Blot, Immunofluorescence

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Downregulation of HMGB1 promoted PCV2 replication. PK-15 cells were transfected with hmgb1-specific RNA interference (RNAi) plasmid (sh-HMGB1) or control RNAi plasmid (sh-NC) for 24 h and then infected with PCV2 (MOI= 1) for 36 h. (A) Effect of hmgb1 knockdown on PCV2 Cap expression (β-actin used as a loading control) as shown by immunoblotting using protein samples from the whole-cell lysates. The gel shown is representative of three independent experiments. (B) The ratios of band intensity of HMGB1 or PCV2 Cap to β-actin (as shown in panel A). (C) Effect of hmgb1 knockdown on PCV2 orf2 (encoding Cap) transcription examined by qPCR using total RNA extracted from the whole-cell lysates. (D) PCV2 replication in hmgb1-silenced cells as assessed by indirect immunofluorescence. Representative fluorescence images are shown (top). Percentage of PCV2-infected cells was calculated by dividing the number of PCV2-infected cells by the total cell number in each group (n = 2 images for each experiment per group) that were counted using ImageJ software. Relative percentages of PCV2-infected cells in the hmgb1-silenced cells are shown with nontransfected but PCV2-infected cells set at 100% (bottom). (E) Effect of hmgb1 silencing on PCV2 genomic DNA copies measured by qPCR using total DNA extracts from whole-cell lysates. Bar charts in panels B, C, D, and E show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Transfection, Plasmid Preparation, Infection, Expressing, Western Blot, Immunofluorescence, Fluorescence, Software

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Nuclear HMGB1 repressed PCV2 replication. PK-15 cells were transfected with recombinant plasmid expressing HMGB1 (pHMGB1) or control plasmid (pFlag) for 24 h and then infected with PCV2 (MOI = 1) for 36 h. Nuclear and cytoplasmic extracts were prepared for immunoblotting as described in the legend for Fig. 1 Immunoblotting of HMGB1 and PCV2 Cap in the nuclear (A) and cytoplasmic (B) fractions. Histone H3 and GAPDH were used as internal controls for nuclear and cytoplasmic extracts, respectively. Representative images from three independent experiments are shown. The ratios of band intensities of HMGB1 or PCV2 Cap to those of histone H3 (as shown in panel A) in the nuclear fraction (C) or to GAPDH (as shown in panel B) in the cytoplasmic fraction (D). (E) Effect of HMGB1 overexpression on PCV2 orf2 transcription in the nuclei examined by qPCR using total RNA extracted from the nuclear fractions. Results were normalized to histone H3 mRNA in the same samples. (F) PCV2 genomic DNA replication in the nuclei of HMGB1-overexpressing cells quantified by qPCR using total DNA extracted from nuclear fractions. Bar charts in panels C to F show means ± SDs from three independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Transfection, Recombinant, Plasmid Preparation, Expressing, Infection, Western Blot, Over Expression

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Ethyl pyruvate inhibited nucleocytoplasmic translocation of HMGB1 in PCV2-infected cells. PK-15 cells were mock infected or infected with PCV2 (MOI = 1) with or without ethyl pyruvate (EP; 7.5 mM) treatment. The cell samples were harvested at 36 hpi. (A) Confocal microscopic images show inhibition of nuclear HMGB1 migration into the cytosol by EP. Cells were immunostained for HMGB1 (green) and PCV2 Cap (red), with nuclei stained with DAPI (blue). Bars, 10 μm. (B) Immunoblotting of HMGB1 and PCV2 Cap in the nuclear and cytoplasmic fractions of PCV2-infected and EP-treated cells. Histone H3 and GAPDH were used as internal controls for nuclear and cytoplasmic extracts, respectively. Representative images from three independent experiments are shown. The ratios of band intensities of HMGB1 or PCV2 Cap to those of histone H3 (as shown in panel B, left) in the nuclear fraction (C) or to GAPDH (as shown in panel B, right) in the cytoplasmic fraction (D). (E) Effect of EP on PCV2 genomic DNA replication by qPCR using DNA extracted from nuclei of PCV2-infected cells treated with 7.5 mM EP. Bar charts in panels C, D, and E show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Translocation Assay, Infection, Inhibition, Migration, Staining, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: N-Acetylcysteine inhibited PCV2-induced HMGB1 translocation from nuclei to cytosol and repressed PCV2 replication. PK-15 cells were mock infected or infected with PCV2 (MOI = 1) for 12 h and then treated with 10 mM N-acetylcysteine (NAC). The cell samples were harvested at 36 hpi. (A) Confocal imaging of HMGB1 distribution in PCV2-infected and NAC-treated cells after the cells were fixed and immunostained for HMGB1 (green) and Cap (red). Nuclei were stained with DAPI (blue). Bars, 10 μm. (B) Blotting of HMGB1 and PCV2 Cap in the nuclear and cytoplasmic extracts of PCV2-infected cells with or without NAC treatment. Histone H3 and GAPDH were used as internal controls for the nuclear and cytoplasmic fractions, respectively. The figure is representative of three independent experiments. The ratios of band intensities of HMGB1 or PCV2 Cap to histone H3 (as shown in panel B, left) in the nuclear fraction (C) or to GAPDH (as shown in panel B, right) in the cytoplasmic fraction (D). (E) Effect of NAC on PCV2 genomic DNA replication by qPCR using DNA extracted from lysates of PCV2-infected cells treated with NAC. (F) Cytosolic ROS levels in PCV2-infected cells with or without treatment by NAC or ethyl pyruvate (EP) as measured by flow cytometry after probing with DCFH-DA. Bar charts in panels C, D, E, and F show means ± SDs from three independent experiments. *, P < 0.05; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Translocation Assay, Infection, Imaging, Staining, Flow Cytometry

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Ethyl pyruvate was inhibitory to PCV2 infection. PK-15 cells were mock infected or infected with PCV2 (MOI = 1) with or without ethyl pyruvate (EP; 7.5 mM) treatment. The cell samples were harvested at 36 hpi. (A) Effect of EP on PCV2 replication in PK-15 cells by immunofluorescence. Percentages of PCV2-infected cells were calculated as described in the legend for Fig. 2. Relative percentages of PCV2-infected cells in the EP-treated cells are shown with untreated but PCV2-infected cells set at 100%. (B) Immunoblotting of HMGB1 and PCV2 Cap in whole-cell lysates with β-actin used as a loading control. (C) The ratios of band intensities of HMGB1 or PCV2 Cap to β-actin (as shown in panel B). Bar charts in panels A and C show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Infection, Immunofluorescence, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: The B box domain of HMGB1 was involved in inhibition of PCV2 replication. (A) Schematic illustration of the full-length and truncated forms of porcine HMGB1 according to its human homolog. All truncated versions, A box, AB box, and B box plus C terminus (B boxCT), were flag tagged. The numbers indicate positions of amino acids. Arrows with C followed by numbers represent key cysteine residues. NLS, nuclear localization signal. PK-15 cells were transfected with recombinant plasmids expressing flag-tagged or full-length HMGB1 for 24 h and then infected with PCV2 (MOI = 1) for 36 h. (B) Numbers of PCV2-infected cells examined by immunofluorescence using anti-Cap monoclonal antibody as the probe (top). Expression of PCV2 Cap and different forms of HMGB1 as assessed by immunoblotting using the whole-cell lysates harvested at 36 hpi and antibodies against Flag, Cap, and HMGB1 (bottom). β-Actin was used as a loading control. The panel B images are representative of three individual experiments. (C) The ratios of band intensities of PCV2 Cap to those of β-actin (as shown at the bottom panel of B). (D) Effect of different HMGB1 truncations on PCV2 DNA replication estimated by qPCR using total DNA extracted from the whole-cell lysate. Bar charts in panels C and D show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Inhibition, Transfection, Recombinant, Expressing, Infection, Immunofluorescence, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: HMGB1 bound to the Ori region of the PCV2 genome. (A) Binding of porcine HMGB1 to PCV2 DNA using the gel shift assay. PCV2 DNA (500 ng) and various concentrations (0 to 5 μg) of purified His-tagged recombinant HMGB1 were mixed in binding buffer. The DNA-protein mixtures were subjected to 0.8% agarose gel electrophoresis to visualize changes of the DNA motility. (B) Binding of HMGB1 to a specific region of PCV2 DNA: full-length and different fragments of PCV2 genome (orf1, orf2, and Ori) were incubated with recombinant HMGB1 protein to identify the region of PCV2 genome involved in HMGB1 binding. (C) To confirm the Ori region is required for HMGB1 binding, the Ori fragment was combined with orf1 or orf2 (Ori-orf1 or Ori-orf2) that were then compared with orf1 or orf2 alone by the gel shift assay. (D) Immunoprecipitation of purified HMGB1 protein (500 μg) and PCV2 DNA (500 ng) mixture by anti-HMGB1 antibody (rabbit IgG as control) and protein A/G agarose. The precipitates were probed with anti-His and anti-HMGB1 antibodies by immunoblotting. (E) Quantification of PCV2 genomic Ori copies by qPCR in DNA extracts from PCV2 DNA-HMGB1 precipitates (shown in panel D) after DNase pretreatment. (F) Blotting of HMGB1 in immunoprecipitates of whole-cell lysates (WCL) of the PK-15 cells infected with PCV2 (36 h) by anti-HMGB1 (rabbit IgG as control) and protein A/G agarose. (G) Quantification of PCV2 genomic Ori copies by qPCR in DNA extracts from immunoprecipitates of whole-cell lysates (shown in panel F) after DNase pretreatment. (H) Blotting of HMGB1 in immunoprecipitates of nuclear extracts of the PK-15 cells infected with PCV2. (I) Quantification of PCV2 genomic Ori copies in the precipitates shown in panel H. Bar charts in panels E, G, and I show means ± SDs from three independent experiments.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Purification, Recombinant, Agarose Gel Electrophoresis, Incubation, Immunoprecipitation, Western Blot, Infection

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Effect of hydrogen peroxide treatment on subcellular localization of HMGB1 and PCV2 replication. (A) H2O2 treatment promoted nucleocytoplasmic translocation of HMGB1. PK-15 cells were treated with or without N-acetylcysteine (NAC; 10 mM) before adding 50 μM H2O2. Cells were fixed and immunostained with anti-HMGB1 (green) for confocal microscopy. Nuclei were labeled with DAPI (blue). (B) Immunoblotting of HMGB1 in the nuclear and cytoplasmic fractions of PK-15 cells treated with H2O2 and NAC. (C) Confocal imaging of PK-15 cells infected by PCV2 with or without 50 μM H2O2 treatment after immunostaining with anti-HMGB1 (green) and anti-Cap (red) antibodies. (D) Blotting of HMGB1 and PCV2 Cap in the nuclear and cytoplasmic extracts of PCV2-infected cells with or without H2O2 treatment. Histone H3 and GAPDH were used as internal controls for the nuclear and cytoplasmic fractions, respectively. (E) Percentages of PCV2-infected cells were calculated from immunofluorescence images as described in the legend for Fig. 2. Relative percentages of PCV2-infected cells in the H2O2-treated cells are shown with nontreated but PCV2-infected cells set at 100%. Bar chart in panel E shows means ± SDs from three independent experiments. **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Translocation Assay, Confocal Microscopy, Labeling, Western Blot, Imaging, Infection, Immunostaining, Immunofluorescence

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Schematic illustration of the interaction between PCV2 and HMGB1 in infected cells. HMGB1 in the nucleus restricts PCV2 replication by binding to the Ori region of the PCV2 genome. PCV2 infection causes increased generation of cellular ROS. Increased ROS promotes nucleocytoplasmic translocation of HMGB1 and lessens sequestration of the viral DNA by HMGB1 in the nucleus, thus enhancing PCV2 replication. N-Acetylcysteine (and probably ethyl pyruvate as well) scavenges PCV2-induced ROS and thus increases retention of HMGB1 in the nucleus, leading to sequestration of viral DNA and reduced PCV2 replication.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Infection, Binding Assay, Translocation Assay

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: TET2 and TET3 associate with the O -GlcNAc transferase OGT and promote GlcNAcylation. ( A ) Silver stain gel of HaloTag-TET protein complex isolations and HaloTag alone control (Ctrl). Protein pulldowns were performed from HEK293T cells overexpressing the indicated HT constructs (see Materials and methods and for details). As not all of the indicated complex isolations were performed at the same time, two separate silver stain gels were run, as shown in this panel. ( B ) Table of transcriptional or chromatin protein interactors found in the various HaloTag-TET isolations. Spectral counts for each interacting protein are shown for biological replicates. TET1, but not TET2, as previously reported ( ; ), shows interaction with SIN3A. OGT interacts with all TET proteins, though it is most highly abundant with TET2 and TET3. ( C ) Detection of OGT by western blotting from HT-TET2 and HT-TET3 pulldowns from ( A ). The indicated pulldowns were probed with an anti-OGT antibody to detect the presence of OGT. OGT and beta-Actin shown as input loading controls. ( D ) TET2 and TET3 co-immunoprecipitate (CoIP) with endogenous OGT from untransfected HEK293T cells. Cell extracts were immunoprecipitated with anti-OGT or rabbit IgG and probed with antibodies against the indicated proteins. An IP control of OGT alone is shown to demonstrate specific capture and enrichment of OGT. Inputs loading controls are shown for all. Note that in this experiment very weak expression of TET2 relative to TET3 is observed. ( E ) The global level of hmC does not change after cell treatment with Alloxan or PUGNAc. Dot blot quantification of global hmC after the indicated treatments. The hmC content is normalized with respect to the input DNA and to mock-treated cells, where the ratio is set at 1.00. Error bars indicate s.d. of three independent experiments. As controls, western blots using anti- O -GlcNAc antibody show the expected decrease in GlcNAcylation with Alloxan and increase with PUGNAc. HDAC1 input loading controls are also shown. Vertical line indicates juxtaposition of lanes non-adjacent within the same blot, exposed for the same time. ( F ) Global decrease in GlcNAcylation is observed in TET2/3 knockdowns. Left: TET2 kd or TET3 kd show decreased GlcNAc activity. Nuclear extracts were prepared from HEK293T cells expressing RNAi Ctrl, RNAi TET2, or RNAi TET3, and UDP-[ 3 H]GlcNAc incorporation was measured. The amount incorporated into the control cells was set at 1. Error bars indicate s.d. of three independent experiments (* P <0.05). Right: Nuclear extracts were prepared from HEK293T cells expressing RNAi Ctrl or RNAi TET2/3 and global GlcNAcylation was visualized with an antibody against O -GlcNAc. HDAC1 input loading control is also shown.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Silver Staining, Control, Construct, Western Blot, Immunoprecipitation, Expressing, Dot Blot, Activity Assay

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: TET2/3–OGT show genomic co-localization around TSSs and impact on H3K4me3 and transcriptional activation. ( A ) Left: Venn diagrams indicating significant overlap of TET2 and OGT bound regions (left part; P -value<10 −10 ) identified after HaloCHIP-Seq in HEK293T cells expressing HT-TET2, or HT-OGT. Right: TET2–OGT targets are primarily found at TSSs and CpG-rich sequences. Similar profiles were also observed for TET3–OGT . ( B ) An analysed subset of TET2–TET3–OGT targets show a lack of DNA methylation and hydroxymethylation, yet display GlcNAcylation. qPCR analysis of TET2–TET3–OGT binding and non-binding regions after MeDIP (top), hMeDIP (middle), or ChIP with an anti- O -GlcNAc antibody (bottom). ‘% Input' represents real-time qPCR values normalized with respect to the input chromatin. Known methylated and hydroxymethylated regions are shown as positive controls in MeDIP and hMeDIP panels.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Activation Assay, Expressing, DNA Methylation Assay, Binding Assay, Methylated DNA Immunoprecipitation, Methylation

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: ( C ) TET2/3–OGT targets in HEK293T cells are enriched for H3K4me3 as depicted in a Venn diagram; P -value<10 −10 . ( D ) Examples of HaloCHIP-Seq OGT, TET2, TET3, and ChIP-Seq H3K4me3 profiles (UCSC tracks). ( E ) Decreased levels of H3K4me3 in TET2 kd cells. Upper-left: decrease in the normalized number of H3K4me3 reads in TET2/3–OGT-binding regions in TET2 kd cells versus control RNAi-treated cells. Upper-right: pie chart showing the percentage of TET2–TET3–OGT binding regions with a statistically significant reduction of the normalized number of H3K4me3 reads for TET2 kd versus control RNAi-treated cells. Lower-part: examples of H3K4me3 ChIP-Seq profiles (UCSC tracks) in TET2–TET3–OGT-binding regions for the RNAi control versus TET2 kd sample. ( F ) Western blot showing global decrease in H3K4me3 in a TET2/3 double kd cells. Lysates from mock HEK293T RNAi kd or TET2/3 kd cells were probed for H3K4me3 using an anti-H3K4 antibody in western blot. Tubulin is shown as a loading control. ( G ) OGT activity is important for H3K4me3. Cell extracts were prepared from HEK293T cells treated with or without the OGT inhibitor Alloxan, and then western blots for H3K4me3 were performed. HDAC1 and H3 are shown as loading controls and a western blot against O -GlcNAc was used to monitor specific GlcNAcylation inhibition by Alloxan. Vertical lines indicate juxtaposition of lanes non-adjacent within the same blot, exposed for the same time. ( H ) Decreases in transcription are observed in both TET2/3 knockdowns and an OGT knockdown. The indicated target genes (which showed decrease in H3K4me3 after TET2 kd; cf. E ) and negative controls (unbound TET2/3–OGT–H3K4me3 targets), were analysed by RT–qPCR in HEK293T cells subjected to the various listed RNAi treatments. Independent experiments were performed in duplicates.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: ChIP-sequencing, Binding Assay, Control, Western Blot, Activity Assay, Inhibition, Knockdown, Quantitative RT-PCR

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: TET2/3 promotes GlcNAcylation of HCF1, and both TET and OGT activity favour the integrity of SET1/COMPASS and SETD1A binding to chromatin. ( A ) Mass spectrometry reveals HCF1, a known target of OGT and component of SET1/COMPASS , as an interacting partner of HT-TET2 and HT-TET3. Biological duplicates and respective spectral counts (SpC) for HCF1 are shown. ( B ) Protein pulldowns of HT-OGT coupled with mass spectrometry identify HCF1, TET2, TET3, and all components of SET1/COMPASS as partners of OGT. Biological duplicates and SpC for each protein identified are shown for HT-OGT and Ctrl isolations. ( C ) The interaction of HCF1 and SET1/COMPASS components with HT-OGT depends on O -GlcNAc activity. Plot showing average SpCs for HCF1 and SET1/COMPASS components isolated from HT-OGT pulldowns of untreated (grey bars) and Alloxan-treated (green bars) HEK293T cells. Error bars represent s.d. of biological duplicates. Representative NSAF plots are shown in . ( D ) The interaction of HT-SETD1A with SET1/COMPASS components and OGT is reduced by a TET2/3 double kd. Plot showing average SpCs for SET1/COMPASS components and OGT isolated from HT-SETD1A pulldowns of control RNAi-treated (grey bars) and TET2/3 kd (blue bars) HEK293T cells. Error bars represent s.d. of biological duplicates. Representative NSAF plots are shown in . ( E ) A significant reduction in HCF1 GlcNAcylation is observed after TET2/3 double kd. Upper diagram shows a schematic representation of full-length HCF1 and its domains . The GlcNAcylated peptides identified by mass spectrometry from HT-SETD1A isolations from control RNAi-treated and TET2/3 kd cells are indicated below. The full-length HCF1 amino-acid sequence (NP_005325.2) shows the corresponding GlcNAcylated peptides highlighted in yellow with RNAi Ctrl on the left and RNAi TET2/3 kd on the right. ( F ) Bioluminescence resonance energy transfer (BRET) assays show reduction of SETD1A binding to histone H3.3 in the presence of an OGT inhibitor and in TET2/3 kd cells. Upper diagram showing the schematic of BRET energy transfer upon binding of a NanoLuc-SETD1A fusion donor and fluorescently labelled Histone H3.3-HaloTag fusion acceptor in live HEK293T cells (see Materials and methods for experimental details and calculation of BRET). Left: BRET measurements were calculated without treatment (grey) or with Alloxan treatment (green). Right: BRET measurement for RNAi control (grey) or RNAi TET2/3 (blue). Biological triplicates ±s.d. are shown.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Activity Assay, Binding Assay, Mass Spectrometry, Isolation, Control, Sequencing, Bioluminescence Resonance Energy Transfer

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: Tet2 knockout mouse tissue shows that Tet2 is needed for global GlcNAcylation and H3K4me3 at target promoters. ( A ) Genome-wide co-localization of endogenous Tet2 with O -GlcNAc and H3K4me3 at promoters and CpG-rich regions. Venn diagrams are shown ( P -value overlap<10 −10 ) as well as the indicated genome-wide distribution. ( B ) Tet2, O -GlcNAc, and H3K4me3 are enriched at many active genes, mirroring the presence of RNA Pol II. Upper panel: Venn diagram showing the overlap of Tet2 and O -GlcNAc with RNA Pol II ( P -value overlap <10 −10 ); lower panel: Box plots showing the reads density at targets and non-targets (others) for ChIP-Seq RNA Pol II or RNA-Seq in mouse bone marrow. ( C ) Global decrease in GlcNAcylation is observed in Tet2 knockout mouse bone marrow. Mouse bone marrow tissues with or without a Tet2 knockout were analysed by western blot for O -GlcNAc levels using an anti- O -GlcNAc antibody. HDAC1 is shown as loading control. ( D ) ChIP-Seq for H3K4me3 in Tet2 knockout mouse tissues shows reduced global H3K4me3 at target promoters. Overall impact on H3K4me3 peak significance (−log( P -value) of the peaks) between wild-type and Tet2 knockout bone marrow is shown. ( E ) Table showing key haematopoietic genes with specifically reduced H3K4me3 in Tet2 knockout as compared to wild type. The location at CpG islands and the promoter class for each is listed. Lower part: example of H3K4me3 ChIP-Seq profiles (UCSC tracks) in wild type versus Tet2 knockout.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Knock-Out, Genome Wide, ChIP-sequencing, RNA Sequencing, Western Blot, Control

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: Model connecting DNA modifying enzymes, TETs, a master cellular sensor protein, OGT, and a histone modifying complex, SET1/COMPASS. Based on our findings, a hierarchical model of the involved proteins, with the cascade of their respective activities, can be envisaged as followed: (1) The first sequence of events in the cascade is the formation of TET2/3–OGT interaction, which promotes OGT GlcNAcylation on numerous proteins, including HCF1; (2) In a TET-dependent manner, a GlcNAcylated HCF1 is important for the formation of the SET1/COMPASS; (3) In the last step, both TET proteins and OGT activity favour binding of SETD1A to chromatin, an event necessary for histone H3K4me3 and subsequent transcriptional activation.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Sequencing, Activity Assay, Binding Assay, Activation Assay

Journal: Molecular Neurobiology

Article Title: TSPYL2 Regulates the Expression of EZH2 Target Genes in Neurons

doi: 10.1007/s12035-018-1238-y

Figure Lengend Snippet: TSPYL2 reduces trimethylation of H3K27 and interacts with EZH2. a Left: representative western blot of hippocampal lysates collected from 2-month-old male littermates. Right: a specific upregulation of H3K27me3 in the mutant hippocampus. Relative protein level to H3 in the wild-type was set as 1. n = 4 per genotype. Error bars represent SEM. * P < 0.05, Student’s t test. b GST-tagged TSPYL2 (GST-L2) pull-down using nuclear lysates from non-transfected (first and bottom rows), Flag-UTX-transfected (second row), and Flag-JMJD3-transfected HEK293 cells (third row). Western blot for proteins indicated on the left. c Components of PRC2 complex (EZH2, EED, SUZ12, RbAp48, and AEBP) were separated by a polyacrylamide gel, probed with recombinant GST, GST-L2, or antibodies to EZH2 as indicated on the top

Article Snippet: For testing the specificities of H3K27me3 antibodies for ChIP (Diagenode, pAb-069-050), antibodies were applied to EpiTitan Histone Peptide Array (Epicypher, #11-2001), washed three times with PBST (PBS pH 7.4, 0.1% Triton-X 100), then hybridized with Alexa Fluor-594 goat anti-rabbit secondary antibodies (Life Technologies), and signals were detected by a Typhoon Scanner (GE Healthcare Life Sciences).

Techniques: Western Blot, Mutagenesis, Transfection, Recombinant

Journal: Molecular Neurobiology

Article Title: TSPYL2 Regulates the Expression of EZH2 Target Genes in Neurons

doi: 10.1007/s12035-018-1238-y

Figure Lengend Snippet: TSPYL2 regulates the level of H3K27me3 in neurons. a Verification of the specificity of H3K27 antibodies for ChIP-seq by hybridization to a histone peptide array. Dotted line divides the sub-arrays. Authentic positive signals appearing in triplets were boxed and peptide identities are 1, H3K27me3 + R36me2; 2, H3K27me3; 3, H4K12ac + K16 ac + H3K27me3. All unmarked triplet peptide signals are from IgG controls to indicate binding of primary antibodies and secondary antibodies. b ChIP-seq results of primary hippocampal neurons at 14 days in culture. H3K27 me3 occupancies at a 10-kb window centering at the TSS were classified into four clusters by k-mean clustering. Metagene analysis shows the increased level of H3K27me3 for cluster1 metagene in Tspyl2 mutant primary neurons. c Selected Gene Ontology of top-enriched biological pathway annotations of cluster 1 genes (DAVID Gene Ontology Bioinformatic Resources, https://david.ncifcrf.gov/home.jsp ). Dotted line, P < 0.05 from P value (a modified Fisher’s exact P value) adjusted by Benjamini-Hochberg correction. d RT-qPCR of example marked genes in cluster 1. Transcript level relative to Hprt and the wild-type level are set as 1. n = 4. Error bars represent SEM. ** P < 0.01, Student t test. e Genome browser views showing ChIP-seq pileups for Gbx2 and Prss16 . Areas of increased H3K27me3 in mutant neurons were boxed. WT: wild-type; Mut: mutant

Article Snippet: For testing the specificities of H3K27me3 antibodies for ChIP (Diagenode, pAb-069-050), antibodies were applied to EpiTitan Histone Peptide Array (Epicypher, #11-2001), washed three times with PBST (PBS pH 7.4, 0.1% Triton-X 100), then hybridized with Alexa Fluor-594 goat anti-rabbit secondary antibodies (Life Technologies), and signals were detected by a Typhoon Scanner (GE Healthcare Life Sciences).

Techniques: ChIP-sequencing, Hybridization, Peptide Microarray, Binding Assay, Mutagenesis, Modification, Quantitative RT-PCR

Journal: Molecular Neurobiology

Article Title: TSPYL2 Regulates the Expression of EZH2 Target Genes in Neurons

doi: 10.1007/s12035-018-1238-y

Figure Lengend Snippet: Gene Ontology analysis of genes with a low level of H3K27me3 in primary neurons. a Selected Gene Ontology of top-enriched biological pathway annotations of cluster 2 H3K27me3 marked genes shows enrichment in processes related to multicellular organism development (DAVID Gene Ontology Bioinformatic Resources). b Cluster 3 marked genes are enriched in G-protein coupled receptor signaling pathway. Dotted line, P < 0.05 from P value (a modified Fisher’s exact P value) adjusted by Benjamini-Hochberg correction. c Distribution of neuronal genes from all RefSeq genes according to the level of H3K27me3. Cluster 1 genes have the highest H3K27me3 level, while cluster 4 genes are unmarked. d Metagene analysis shows a peak of H3K27me3 at TSS for cluster 2 neuronal genes, which is reduced in Tspyl2 mutant primary neurons

Article Snippet: For testing the specificities of H3K27me3 antibodies for ChIP (Diagenode, pAb-069-050), antibodies were applied to EpiTitan Histone Peptide Array (Epicypher, #11-2001), washed three times with PBST (PBS pH 7.4, 0.1% Triton-X 100), then hybridized with Alexa Fluor-594 goat anti-rabbit secondary antibodies (Life Technologies), and signals were detected by a Typhoon Scanner (GE Healthcare Life Sciences).

Techniques: Modification, Mutagenesis

Journal: Molecular Neurobiology

Article Title: TSPYL2 Regulates the Expression of EZH2 Target Genes in Neurons

doi: 10.1007/s12035-018-1238-y

Figure Lengend Snippet: Loss of TSPYL2 reduces expression of specific H3K27me3 marked neuronal genes. a H3K27me3 and EZH2 ChIP-seq signals around TSS of selected genes important for neuron functions. b RT-qPCR showing significantly reduced expression of mainly cluster 2 neuronal genes in DIV14 mutant neurons. Transcript level relative to Hprt and the wild-type level was used for normalization. Error bars represent SEM. * P < 0.05, ** P < 0.01, Student’s t test. WT: wild-type, n = 5–7; Mut: mutant, n = 5–11

Article Snippet: For testing the specificities of H3K27me3 antibodies for ChIP (Diagenode, pAb-069-050), antibodies were applied to EpiTitan Histone Peptide Array (Epicypher, #11-2001), washed three times with PBST (PBS pH 7.4, 0.1% Triton-X 100), then hybridized with Alexa Fluor-594 goat anti-rabbit secondary antibodies (Life Technologies), and signals were detected by a Typhoon Scanner (GE Healthcare Life Sciences).

Techniques: Expressing, ChIP-sequencing, Quantitative RT-PCR, Mutagenesis

Journal: Science Advances

Article Title: Dedifferentiation and neuronal repression define familial Alzheimer’s disease

doi: 10.1126/sciadv.aba5933

Figure Lengend Snippet: ( A ) TSS heatmap coverage plots of H3K4Me3 and H3K27Me3 in NDC and PSEN1 M146L and PSEN1 A246E hiPSC-derived neurons as determined by ChIP-seq. ( B ) Annotation and directionality of differential ChIP-seq peaks in PSEN1 M146L and PSEN1 A246E hiPSC-derived neurons relative to NDC. ( C ) TF motif enrichment of regions with an increase in activating histone methylation status (increase in H3K4Me3 or decrease in H3K27Me3; top) or an increased in repressive histone methylation status (decrease in H3K4Me3 or increase in H3K27Me3; bottom) occurring within promoter or TSS-associated enhancer regions using GimmeMotifs and the HOCOMOCOv11 motif database. ( D and E ) chipenrich enrichment analysis of regions with an increase in activating histone methylation status (increase in H3K4Me3 or decrease in H3K27Me3; top) or an increased in repressive histone methylation status (decrease in H3K4Me3 or increase in H3K27Me3; bottom) occurring within promoter or TSS-associated enhancer regions using (D) ENCODE-ChEA consensus and ReMap TF-gene target databases or (E) GOBP and Hallmark ontology databases. ( F ) Venn-Euler diagram of overlap of DEGs with regions of differential H3K4Me3 or H3K27Me3 occurring within promoter or TSS-associated enhancer regions in PSEN1 M146L (top) and PSEN1 A246E (bottom) hiPSC-derived neurons. ( G ) Histone methylation density in PSEN1 M146L around DEGs with corresponding gain or loss of H3K4Me3 or H3K27Me3 status within promoter and enhancer regions. ( H and I ) Commonly enriched terms by hypergeometric (hg) enrichment analysis using the (H) ENCODE-ChEA Consensus, ReMap, Transfac/JASPAR (T/J), and TRRUST (T) TF-gene target databases or (I) GOBP and Hallmark databases for genes with directionally correlated increased gene expression and gain of activating H3KMe3 status (top) or decreased gene expression and gain of repressive H3KMe3 status (bottom).

Article Snippet: Five hundred nanograms of purified, sonicated chromatin was incubated with 5 μl of H3K4Me3 (Active Motif, catalog no. 39159) or H3K27Me3 (Active Motif, catalog no. 39155) rabbit polyclonal antibody overnight at 4°C.

Techniques: Derivative Assay, ChIP-sequencing, Methylation, Gene Expression

Journal: Science Advances

Article Title: Dedifferentiation and neuronal repression define familial Alzheimer’s disease

doi: 10.1126/sciadv.aba5933

Figure Lengend Snippet: ( A ) Venn diagram of overlap of DEGs, which have intersecting and directionally corresponding change in H3K4Me3 or H3K27Me3 status flanking regions of differential accessibility occurring within gene promoters or TSS-associated enhancers in PSEN1 M146L hiPSC-derived neurons. ( B and C ) Hypergeometric enrichment analysis using the (B) ENCODE-ChEA consensus and ReMap TF-gene target databases or (C) GOBP and Hallmark databases for genes with increased gene expression, chromatin accessibility, and gain of activating H3KMe3 status (top; yellow) or decreased gene expression, chromatin accessibility, and gain of repressive H3KMe3 status (bottom; blue). ( D to F ) ATAC-seq, H3K4Me3, and H3K27Me3 read profiles around the promoter regions of (D) nonectoderm lineage–controlling TFs with increased gene expression, (E) neuronal lineage– and function-controlling TFs with decreased gene expression, or (F) lineage-defining miRNAs with decreased expression.

Article Snippet: Five hundred nanograms of purified, sonicated chromatin was incubated with 5 μl of H3K4Me3 (Active Motif, catalog no. 39159) or H3K27Me3 (Active Motif, catalog no. 39155) rabbit polyclonal antibody overnight at 4°C.

Techniques: Derivative Assay, Gene Expression, Expressing

Journal: Nucleic Acids Research

Article Title: Transcriptional induction by ecdysone in Drosophila salivary glands involves an increase in chromatin accessibility and acetylation

doi: 10.1093/nar/gkaf284

Figure Lengend Snippet: E23 expression in the salivary glands suppresses ‘active’ ESEs co-bound with EcR and CBP/Nejire at 20E-activated loci. ( A ) Venn diagram reflecting an intersection of EcR and CBP/Nejire peaks located in 20E-activated loci ±5 kb from a total number of EcR and CBP/Nejire peaks in these loci defined by ChIP-Seqs in salivary glands of hsp-e23 wandering larvae in untreated conditions. Regions co-bound with CBP/Nejire and EcR were termed ESEs. ( B ) A scheme displaying two principles of classification of ESEs. Location: ESEs located in ±250 bp of TSSs were defined as proximal ESEs, ESEs located outside of the TSSs but within gene loci, ±5 kb were called distal ESEs. Activity: ESEs are categorized based on their levels of H3K27Ac acetylation. Those with high levels of H3K27Ac are considered ‘active’. ESEs with lower levels of H3K27Ac are ‘poised’. Hierarchical clustering was used to separate ESEs into groups based on their activity levels (provided in ). Created in BioRender ( https://BioRender.com/0ngjjab ). ( C ) Average distribution of EcR, CBP, FAIRE, H3K27Ac enrichment estimated by ChIP-Seqs at ‘active’ proximal (located ±250 bp of TSSs, N = 64) and active distal (located outside of the TSSs but within gene loci, ±5 kb, N = 258) ESEs in 20E-activated primary loci in salivary glands in control condition (NHS) and after treatment of hsp-e23 larvae 20–22 h before pupariation with double 1-h heat shock (with a 1-h rest at RT) (HS). ( D ) Average distribution of EcR, CBP, FAIRE, H3K27Ac proteins estimated by ChIP-Seqs at ‘poised’ proximal (located ±250 bp of TSSs, N = 135) and ‘poised’ distal (located outside of the TSSs but within gene loci, ±5 kb, N = 369) ESEs in 20E-activated primary loci in salivary glands in control condition (NHS) and after treatment of hsp-e23 larvae 20–22 h before pupariation with double 1-h heat shock (with a 1-h rest at RT) (HS). For panels (C) and (D) ChIP-Seq binding levels were calculated as a ratio to Input (for FAIRE and H3K27Ac the Input was subtracted from sample). The X-axis represents the distance to the ESE in kbp. Average profiles were calculated as a median of binding level with the standard error displayed on the profiles. The FC was calculated using normalized coverage within 500 bp around the summit peak for EcR, CBP, FAIRE and within 1000 bp around the summit peak for H3K27Ac of the analysed ESEs (as a ratio of NHS signal to HS signal). The results of the paired t -test analysis are provided on the graphs, where ‘**’ means P ≤ .01. ( E ) Scheme displaying that the transcriptional response to 20E in salivary glands involves activating a subset of ESEs with high H3K27Ac levels, highlighting the importance of 20E in regulating chromatin state and EcR binding dynamics. Created in BioRender ( https://BioRender.com/n650dzg ).

Article Snippet: Antibodies against histone H3K27Ac (39133) were purchased in Active motif.

Techniques: Expressing, Activity Assay, Control, ChIP-sequencing, Binding Assay

Journal: Nucleic Acids Research

Article Title: Transcriptional induction by ecdysone in Drosophila salivary glands involves an increase in chromatin accessibility and acetylation

doi: 10.1093/nar/gkaf284

Figure Lengend Snippet: ‘Active’ ESEs, sensitive to 20E depletion, are tissue-specific, as evidenced by our and previously published data. Average distribution of EcR, CBP, FAIRE and H3K27Ac enrichment estimated by ChIP-Seqs at ( A ) ‘active’ proximal (located ±250 bp of TSSs, N = 64) ESEs and ( B ) ‘active’ distal (located outside of the TSSs but within gene loci, ±5 kb, N = 258) ESEs in salivary glands (SG) and brain (BR) of hsp-e23 wandering larva in untreated conditions. ChIP-Seq binding level was calculated as a ratio to Input. For FAIRE-Seq and H3K27Ac the Input was subtracted from sample. The X-axis represents the distance to the ESE in kbp. Average profiles were calculated as a median of binding level with the standard error displayed on the profiles. The FC was calculated using normalized coverage within 500 bp around the summit peak for EcR, CBP, FAIRE and within 1000 bp around the summit peak for H3K27Ac of the analysed ESEs (as a ratio of SG signal to a BR signal). The results of the paired t -test analysis are provided on the graphs, where ‘**’ means P ≤ .01. ( C ) Average FAIRE-Seq signals from the previously published data (from ) at distal (N = 258) and proximal (N = 64) ‘active’ ESEs of 20E-activated targets in salivary glands. The FC was calculated using normalized coverage within 500 bp around the summit peak of the analysed ESEs (as a ratio of SG signal to Wing and Leg signals). The FC for proximal ‘active’ loci were 5.1 for SG/Wing and 4.0 for SG/Leg. The FC for distal ‘active’ loci were 9.7 for SG/Wing and 13.2 for SG/Leg. The results of the paired t -test analysis are provided on the graphs, where ‘**’ means P ≤ .01. ( D ) PCA plot represents a strong correlation between the FAIRE-Seq signals for the salivary glands obtained previously and in a current study. In other tissues the chromatin accessibility levels at the described ESEs varied. For the PCA analysis FAIRE-Seq signals were calculated using normalized coverage within 500 bp around the summit peak of the analysed ESEs. ( E ) Model illustrating the regulation of 20E-activated target loci in salivary glands. We suggest that 20E-activated targets are regulated by a tissue-specific set of ‘active’ ESEs. In salivary glands, these ‘active’ ESEs have CBP/Nejire and EcR peaks, as well as increased chromatin accessibility and acetylation, whereas in the brain, they are silent. Created in BioRender ( https://BioRender.com/okxy4sl ).

Article Snippet: Antibodies against histone H3K27Ac (39133) were purchased in Active motif.

Techniques: ChIP-sequencing, Binding Assay

Journal: Molecular cell

Article Title: The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

doi: 10.1016/j.molcel.2021.03.034

Figure Lengend Snippet: (A) Top: Venn diagram depicting differentially expressed genes (nascent RNA-seq, log2 fold-change >2 and FDR <0.05) in Thap1−/− versus WT and Thap1−/−Brca1Δ11 versus Brca1Δ11 MEFs in relation to THAP1-bound genes (ChIP-seq). The number of genes that were shown to be bound by THAP1 and were either downregulated or upregulated in THAP1-deficient MEFs are shown in blue and red, respectively.

Article Snippet: ChIP-seq was performed as described previously ( Shinoda et al., 2019 ) with a rabbit polyclonal antibody against THAP1 (Proteintech, 12584–1-AP).

Techniques: RNA Sequencing, ChIP-sequencing

Journal: Molecular cell

Article Title: The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

doi: 10.1016/j.molcel.2021.03.034

Figure Lengend Snippet: (A) Western blot analysis of doxycycline-dependent expression of exogenous SHLD1 (left) and THAP1 (right) proteins in WT MEFs 24 to 96 hours after induction with doxycycline (Dox) as detected by anti-Flag antibody.

Article Snippet: ChIP-seq was performed as described previously ( Shinoda et al., 2019 ) with a rabbit polyclonal antibody against THAP1 (Proteintech, 12584–1-AP).

Techniques: Western Blot, Expressing

Journal: Molecular cell

Article Title: The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

doi: 10.1016/j.molcel.2021.03.034

Figure Lengend Snippet: (A-B) Quantification of RPA2 (A) and RAD51 (B) foci in individual EdU-positive (S-phase) nuclei of WT, Brca1Δ11, Trp53bp1−/−Brca1Δ11 and two individual clones of Thap1−/− Brca1Δ11 MEFs. Cells were irradiated with 10 Gy and analyzed 4 h post-IR. Statistical significance was determined by Welch’s t-test.

Article Snippet: ChIP-seq was performed as described previously ( Shinoda et al., 2019 ) with a rabbit polyclonal antibody against THAP1 (Proteintech, 12584–1-AP).

Techniques: Clone Assay, Irradiation

Journal: Molecular cell

Article Title: The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

doi: 10.1016/j.molcel.2021.03.034

Figure Lengend Snippet: (A) Representative flow cytometry plots of IgM-to-IgA class switch recombination (CSR) in WT, Trp53bp1−/−, Shld1−/−, Shld3−/− and two individual clones of Thap1−/− (#4 and #12) CH12-F3 cells 24 hours after cytokine stimulation (IL-4, CD40L and TGFβ). Unstimulated WT cells are shown as a negative control. Quantification of IgM-to-IgA CSR is shown on the right and represents mean ± s.d., n=3.

Article Snippet: ChIP-seq was performed as described previously ( Shinoda et al., 2019 ) with a rabbit polyclonal antibody against THAP1 (Proteintech, 12584–1-AP).

Techniques: Flow Cytometry, Clone Assay, Negative Control

Journal: Molecular cell

Article Title: The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

doi: 10.1016/j.molcel.2021.03.034

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ChIP-seq was performed as described previously ( Shinoda et al., 2019 ) with a rabbit polyclonal antibody against THAP1 (Proteintech, 12584–1-AP).

Techniques: Purification, Blocking Assay, Virus, Bacteria, Expressing, CRISPR, Knock-Out, Recombinant, Transfection, Cloning, PCR Cloning, Protease Inhibitor, Ligation, Library Quantification, Selection, Flow Cytometry, Cell Viability Assay, cDNA Synthesis, SYBR Green Assay, Cell Culture, Mutagenesis, Illumina Sequencing, Software, Microscopy, Imaging, Irradiation

Journal: Nature Communications

Article Title: Nuclear ADP-ribosylation drives IFNγ-dependent STAT1α enhancer formation in macrophages

doi: 10.1038/s41467-021-24225-2

Figure Lengend Snippet: a Heat map of ChIP-seq data representing STAT1α binding in BMDMs treated with IFNγ ± PJ34. BMDMs were treated with IFNγ for 1 h and ChIP-seq was performed using STAT1 antibody. Enrichment of peaks is shown relative to the untreated control. b , c Box plots ( b ) and browser tracks ( c ) representing ‘maintained,’ ‘depleted,’ and ‘gained’ STAT1α peaks from ChIP-seq data. A cutoff of 1x MAD (median absolute deviation) was used to define ‘gained’ and ‘depleted’ peaks. ‘Maintained’ peaks were defined with a cutoff of 0.5x MAD (Wilcoxon Signed-Rank test; p < 2.2 × 10 -16 ). Number of peaks for box plots was indicated in ( a ). Boxes represent 25 th –75 th percentile (line at median) with whiskers at 1.5*IQR. d A schematic diagram showing the integration of ChIP-seq data with RNA-seq to correlate STAT1α binding with changes in gene expression in BMDMs. e PARP-1-dependent changes in STAT1α binding correlate with altered transcriptional outcomes. The nearest neighbor gene expression for each category of STAT1α peaks was calculated as shown in ( d ). The line plots represent the fold change in gene expression upon IFNγ treatment ± PJ34 from the RNA-seq assays shown in Fig. . The mRNA levels are expressed as fold change over the untreated control.

Article Snippet: The following antibodies were used for immunoblotting and immunofluorescent staining: STAT1 rabbit polyclonal antibody (Cell Signaling, 9172 L); Phospho-STAT1α (Ser727) rabbit monoclonal antibody (Cell Signaling, 8826 S); Phospho-STAT1α (Ser727) rabbit polyclonal antibody (Cell Signaling Technologies, 9177); Phospho-STAT1α (Tyr701) rabbit polyclonal antibody (Cell Signaling Technologies, 9167); Flag mouse monoclonal antibody (Sigma-Aldrich, F3165); β-tubulin rabbit polyclonal antibody (Abcam, ab6046); p300 mouse monoclonal antibody (Active motif, 61401); Acetyl-CBP (Lys1535)/p300 (Lys1499) rabbit polyclonal antibody (Cell Signaling, 4771); rabbit IgG (ThermoFisher Scientific, 10500 C); goat anti-rabbit HRP-conjugated IgG (Pierce, 31460); and goat anti-mouse HRP-conjugated IgG (Pierce, 31430).

Techniques: ChIP-sequencing, Binding Assay, Control, RNA Sequencing, Gene Expression

Journal: Nature Communications

Article Title: Nuclear ADP-ribosylation drives IFNγ-dependent STAT1α enhancer formation in macrophages

doi: 10.1038/s41467-021-24225-2

Figure Lengend Snippet: a PARP-1 deletion attenuates STAT1α phosphorylation at S727. Immunofluorescent staining for phospho-STAT1α (S727p) was performed in BMDMs collected from wild-type ( Parp1 +/+ ) or Parp1 knockout ( Parp1 -/- ) mice treated with IFNγ (1 h). Nuclei were visualized with DAPI staining. Scale bar: 10 μm. b Violin plots showing quantification of the immunofluorescence data from ( a ) in BMDMs from Parp1 +/+ ( n = 3) and Parp1 -/- ( n = 3) mice (one-way ANOVA followed by Tukey’s multiple comparison tests; **** < 0.0001; n.s., not significant at 0.05). c Immunoblots showing the relative levels of STAT1α S727p, STAT1α Y701p, total STAT1α and Tubulin in BMDMs from Parp1 +/+ and Parp1 -/- mice. Rep1 and Rep2 represent two independent biological replicates. Uncropped immunoblots are provided as a Source Data file. d Inhibition of PARP-1 catalytic activity by PJ34 blocks STAT1α S727 phosphorylation. Immunofluorescent staining was performed as in ( a ). BMDMs were treated with IFNγ (1 h) ± PJ34. Nuclei were visualized with DAPI staining. Scale bar: 10 μm. e Violin plots showing quantification of the immunofluorescence data from ( d ) for BMDMs from 3 mice for each treatment (one-way ANOVA followed by Tukey’s multiple comparison test; *** < 0.0001). f Inhibition of PARP-1 catalytic activity by PJ34 results in reduced enrichment of H3K27ac (right) levels at maintained STAT1α binding sites (left) . ChIP-seq for STAT1α and H3K27ac was carried out in BMDMs treated with IFNγ (1 h) ± PJ34 ( n = 252 peaks; Wilcoxon Signed-Rank test; p < 2.2 × 10 -16 ). Boxes represent 25 th –75 th percentile (line at median) with whiskers at 1.5*IQR.

Article Snippet: The following antibodies were used for immunoblotting and immunofluorescent staining: STAT1 rabbit polyclonal antibody (Cell Signaling, 9172 L); Phospho-STAT1α (Ser727) rabbit monoclonal antibody (Cell Signaling, 8826 S); Phospho-STAT1α (Ser727) rabbit polyclonal antibody (Cell Signaling Technologies, 9177); Phospho-STAT1α (Tyr701) rabbit polyclonal antibody (Cell Signaling Technologies, 9167); Flag mouse monoclonal antibody (Sigma-Aldrich, F3165); β-tubulin rabbit polyclonal antibody (Abcam, ab6046); p300 mouse monoclonal antibody (Active motif, 61401); Acetyl-CBP (Lys1535)/p300 (Lys1499) rabbit polyclonal antibody (Cell Signaling, 4771); rabbit IgG (ThermoFisher Scientific, 10500 C); goat anti-rabbit HRP-conjugated IgG (Pierce, 31460); and goat anti-mouse HRP-conjugated IgG (Pierce, 31430).

Techniques: Phospho-proteomics, Staining, Knock-Out, Immunofluorescence, Comparison, Western Blot, Inhibition, Activity Assay, Binding Assay, ChIP-sequencing

Journal: Nature Communications

Article Title: Nuclear ADP-ribosylation drives IFNγ-dependent STAT1α enhancer formation in macrophages

doi: 10.1038/s41467-021-24225-2

Figure Lengend Snippet: a STAT1α is ADPRylated in cells. Immunoblots showing ADPRylation of STAT1α in immortalized BMDMs (iBMDMs). Flag-tagged STAT1α was ectopically expressed in iBMDMs and immunoprecipitated using a Flag antibody. Flag-tagged GFP was used as a vector control. PAR levels were detected using an ADP-ribose detection reagent (WWE-Fc reagent). The immunoblots are representative of 3 independent experiments. Uncropped immunoblots are provided as a Source Data file. b Immunoblots showing ADPRylation of STAT1α by PARP-1 in vitro. In vitro ADPRylation reactions were setup as indicated. Recombinant PARP-1 and STAT1α expressed and purified from Sf9 insect cells were incubated with 100 μM NAD + . The immunoblots are representative of 3 independent experiments. Uncropped immunoblots are provided as a Source Data file. c Schematic representation of the protocol used for determining the sites of ADPRylation on STAT1α using mass spectrometry. d Schematic representation showing the sites of ADPRylation on STAT1α determined by mass spectrometry. ADPRylated glutamate and aspartate residues on STAT1α are indicated by blue circles and sites of phosphorylation are indicated by red circles. e Mutation of mass spectrometry-identified ADPRylation sites inhibits ADPRylation on STAT1α in IFNγ-treated iBMDMs. Mutations of the amino acids shown in ( d ) were engineered into full-length STAT1α. E393/4Q and D721N are indicated as DBDmut and TAmut, respectively. iBMDM cells were incubated with 250 μM NAD + for the ADPRylation reactions in nuclei. Immunoblotting was performed as in ( a ). Uncropped immunoblots are provided as a Source Data file. f Nitric oxide synthase (NOS) activity assay measuring relative NOS levels in iBMDMs expressing Wt vs. ADPRylation-deficient STAT1α mutants. iBMDM cells were treated with IFNγ for 24 h ( n = 3; Student’s two-tailed, unpaired t-test * = 0.0208 for Wt vs. DBDmut; * = 0.0410 for Wt vs. TAmut). Error bars represent mean ± SEM. g Loss of site-specific ADPRylation on the STAT1α DBD or TA domain results in reduced phagocytotic capacity in macrophages. Phagocytosis in iBMDMs was assayed using S. aureus bioparticles conjugated to pHrodo-green. The images are representative of 3 independent experiments. Scale bar: 44 μm. h , i Site-specific ADPRylation of STAT1α on its DBD or TA domain is required for IFNγ-stimulated increases in cellular glycolysis. h Glycolytic rate profile of iBMDMs expressing Wt or ADPRylation-deficient STAT1α mutants using Seahorse assays ( n = 3). Error bars represent mean ± SEM. i Fold change in the amount of basal glycolysis observed upon IFNγ treatment ( n = 3; two-tailed, unpaired t-test * = 0.0268 for Wtvs. DBDmut; * = 0.0207 for Wt vs. TAmut). Error bars represent mean ± SEM.

Article Snippet: The following antibodies were used for immunoblotting and immunofluorescent staining: STAT1 rabbit polyclonal antibody (Cell Signaling, 9172 L); Phospho-STAT1α (Ser727) rabbit monoclonal antibody (Cell Signaling, 8826 S); Phospho-STAT1α (Ser727) rabbit polyclonal antibody (Cell Signaling Technologies, 9177); Phospho-STAT1α (Tyr701) rabbit polyclonal antibody (Cell Signaling Technologies, 9167); Flag mouse monoclonal antibody (Sigma-Aldrich, F3165); β-tubulin rabbit polyclonal antibody (Abcam, ab6046); p300 mouse monoclonal antibody (Active motif, 61401); Acetyl-CBP (Lys1535)/p300 (Lys1499) rabbit polyclonal antibody (Cell Signaling, 4771); rabbit IgG (ThermoFisher Scientific, 10500 C); goat anti-rabbit HRP-conjugated IgG (Pierce, 31460); and goat anti-mouse HRP-conjugated IgG (Pierce, 31430).

Techniques: Western Blot, Immunoprecipitation, Plasmid Preparation, Control, In Vitro, Recombinant, Purification, Incubation, Mass Spectrometry, Phospho-proteomics, Mutagenesis, Nos Activity Assay, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Nuclear ADP-ribosylation drives IFNγ-dependent STAT1α enhancer formation in macrophages

doi: 10.1038/s41467-021-24225-2

Figure Lengend Snippet: a Structure of DNA-bound STAT1α showing sites of ADPRylation on the DBD. STAT1α is shown in pink and DNA is shown in blue. ADPRylated residues (E393 and E394) are highlighted in red in the expanded view. The structure is from Protein Data Bank (PDB) 1BF5. b Heatmap of ChIP-seq data showing STAT1α enrichment in the top 50% of ‘gained’ STAT1α peaks in iBMDMs expressing wild-type (Wt) or DBD mutant (DBDmut) STAT1α with concurrent shRNA-mediated knockdown of endogenous STAT1α. The cells were treated ± IFNγ for 1 h. ‘Gained’ peaks were defined using a 4x MAD cutoff. c , d Browser tracks ( c ) and box plots ( d ) of ChIP-seq data representing ‘gained’ STAT1α peaks in iBMDMs expressing DBDmut compared to iBMDMs expressing Wt STAT1α ( n = 227 peaks; Wilcoxon Signed-Rank test; p < 2.2 × 10 -16 ). Boxes represent 25 th –75 th percentile (line at median) with whiskers at 1.5*IQR. e Motifs enriched at gained STAT1α binding sites (DBDmut relative to Wt STAT1α). De novo motif analysis was performed using MEME. The predicted motifs were matched to known motifs using TOMTOM. P-values were generated using default parameters in TOMTOM (see Methods). f Binding of STAT1α DBDmut to non-consensus motifs. STAT1α from iBMDMs expressing Wt or DBDmut was incubated with double-stranded DNA oligonucleotides containing a consensus STAT3 binding sequence. Bound material and input were analyzed by immunoblotting for STAT1α. Uncropped immunoblots are provided as a Source Data file. g Gene expression associated with gained STAT1α peaks. Line plots representing fold change in nearest neighbor gene expression upon IFNγ treatment from RNA-seq in iBMDMs expressing Wt or DBDmut STAT1α with concurrent shRNA-mediated knockdown of endogenous STAT1α. h Line plots representing fold change in IFNγ-stimulated gene expression in iBMDMs expressing Wt or DBDmut STAT1α relative to an untreated control.

Article Snippet: The following antibodies were used for immunoblotting and immunofluorescent staining: STAT1 rabbit polyclonal antibody (Cell Signaling, 9172 L); Phospho-STAT1α (Ser727) rabbit monoclonal antibody (Cell Signaling, 8826 S); Phospho-STAT1α (Ser727) rabbit polyclonal antibody (Cell Signaling Technologies, 9177); Phospho-STAT1α (Tyr701) rabbit polyclonal antibody (Cell Signaling Technologies, 9167); Flag mouse monoclonal antibody (Sigma-Aldrich, F3165); β-tubulin rabbit polyclonal antibody (Abcam, ab6046); p300 mouse monoclonal antibody (Active motif, 61401); Acetyl-CBP (Lys1535)/p300 (Lys1499) rabbit polyclonal antibody (Cell Signaling, 4771); rabbit IgG (ThermoFisher Scientific, 10500 C); goat anti-rabbit HRP-conjugated IgG (Pierce, 31460); and goat anti-mouse HRP-conjugated IgG (Pierce, 31430).

Techniques: ChIP-sequencing, Expressing, Mutagenesis, shRNA, Knockdown, Binding Assay, Generated, Incubation, Sequencing, Western Blot, Gene Expression, RNA Sequencing, Control

Journal: Nature Communications

Article Title: Nuclear ADP-ribosylation drives IFNγ-dependent STAT1α enhancer formation in macrophages

doi: 10.1038/s41467-021-24225-2

Figure Lengend Snippet: a Amino acid sequence showing the ADPRylation (blue) and phosphorylation (red) sites in the TA domain of STAT1α. b ADPRylation at D721 in the TA domain of STAT1α is required for IFNγ-dependent phosphorylation. Immunoblots showing total STAT1α, STAT1α S727p and STAT1α Y701p from iBMDMs expressing Wt, DBDmut, or TAmut STAT1α. Uncropped immunoblots are provided as a Source Data file. c ADPRylation of STAT1α stimulates p300 autoacetylation. Immunoblots showing the acetylation of p300 from in vitro reactions performed in the presence of ADPRylated STAT1α under the conditions indicated. The immunoblots are representative of 3 independent experiments. Uncropped immunoblots are provided as a Source Data file. d ADPRylation of STAT1α on its TA domain is required for p300 autoacetylation. Immunoblots show autoacetylation of p300 in the presence of STAT1α Wt, DBDmut or TAmut from ADPRylation reactions with PARP-1 as indicated. The immunoblots are representative of 3 independent experiments. Uncropped immunoblots are provided as a Source Data file. e Loss of ADPRylation on the STAT1α TA domain results in reduced H3K27ac levels at maintained STAT1α binding sites. Box plots of ChIP-seq data showing STAT1α and H3K27ac enrichment in Wt- or TAmut-expressing iBMDMs ( n = 492 peaks; Wilcoxon Signed-Rank test; p < 2.2 × 10 -16 ). Boxes represent 25 th –75 th percentile (line at median) with whiskers at 1.5*IQR. The cells were treated with IFNγ for 1 h. f Line plots representing fold change in IFNγ-stimulated gene expression in iBMDMs expressing Wt or TAmut STAT1α relative to an untreated control. The iBMDMs ectopically expressing Wt or TAmut STAT1α had concurrent shRNA-mediated knockdown of endogenous STAT1α. g Model showing the regulation of pro-inflammatory responses in macrophages by PARP-1-mediated site-specific ADPRylation of STAT1α. See the text for details.

Article Snippet: The following antibodies were used for immunoblotting and immunofluorescent staining: STAT1 rabbit polyclonal antibody (Cell Signaling, 9172 L); Phospho-STAT1α (Ser727) rabbit monoclonal antibody (Cell Signaling, 8826 S); Phospho-STAT1α (Ser727) rabbit polyclonal antibody (Cell Signaling Technologies, 9177); Phospho-STAT1α (Tyr701) rabbit polyclonal antibody (Cell Signaling Technologies, 9167); Flag mouse monoclonal antibody (Sigma-Aldrich, F3165); β-tubulin rabbit polyclonal antibody (Abcam, ab6046); p300 mouse monoclonal antibody (Active motif, 61401); Acetyl-CBP (Lys1535)/p300 (Lys1499) rabbit polyclonal antibody (Cell Signaling, 4771); rabbit IgG (ThermoFisher Scientific, 10500 C); goat anti-rabbit HRP-conjugated IgG (Pierce, 31460); and goat anti-mouse HRP-conjugated IgG (Pierce, 31430).

Techniques: Sequencing, Phospho-proteomics, Western Blot, Expressing, In Vitro, Binding Assay, ChIP-sequencing, Gene Expression, Control, shRNA, Knockdown