Journal: eLife

Article Title: SIRT2 deacetylase regulates the activity of GSK3 isoforms independent of inhibitory phosphorylation

doi: 10.7554/eLife.32952

Figure Lengend Snippet: ( A ) Scatter plot showing cardiac hypertrophy, as measured by Heart weight/Tibia Length (HW/TL) ratio of 8 weeks old 129/Sv mice treated with either vehicle or isoproterenol (ISO) at the dose of 10 mg/kg/day. ISO was continuously infused for 7 days using osmotic mini-pumps. n = 9–10 mice per group. Data is presented as mean ± s.d, *p<0.05. Student’s t test was used to calculate the p values. ( B ) Scatter plot representing left ventricular posterior wall thickness of 8 weeks old 129/Sv mice treated with either vehicle or ISO at the dose of 10 mg/kg/day. ISO was continuously infused for 7 days using osmotic mini-pumps. n = 6 mice per group. Data is presented as mean ± s.d, *p<0.05. Student’s t test was used to calculate the p values. ( C ) Scatter plot indicating the contractile functions of heart as represented by ejection fraction of 8 weeks old 129/Sv mice treated with either vehicle or ISO at the dose of 10 mg/kg/day. ISO was continuously infused for 7 days using osmotic mini-pumps. n = 6 mice per group. Data is presented as mean ± s.d, *p<0.05. Student’s t test was used to calculate the p values. ( D ) Histogram showing GSK3β activity assay in heart lysates of vehicle or ISO-treated 8 weeks old 129/Sv mice. Mice were treated with either vehicle or ISO at the dose of 10 mg/kg/day for 7 days using osmotic mini-pumps. GSK3β was immunoprecipitated from the heart lysates of vehicle or ISO infused mice using anti-GSK3β antibody, clone GSK-4B (Sigma). The immunoprecipitated GSK3β was incubated with the peptide substrate in the presence of γ− 32 P-ATP. The incorporation of 32 P into the GSK3β peptide substrate, which contains specific phosphorylation residues of GSK3β was measured. n = 10 mice per group. Data is presented as mean ± s.d, *p<0.05. Student’s t test was used to calculate the p values. ( E ) Eight weeks old 129/Sv mice were treated with either vehicle or ISO at the dose of 10 mg/kg/day for 7 days using osmotic mini-pumps. GSK3β was immunoprecipitated from the heart lysates of vehicle or ISO infused mice using anti-GSK3β antibody (sc-9166, Santa Cruz Biotechnolgy) and the affinity resin immobilized with protein A/G. Western blotting analysis was performed to detect the levels of GSK3β acetylation (Ac-Lys) by anti-acetyl-lysine antibody. IgG was used as negative control in this assay. Heart tissue lysates (WCL) were probed for indicated proteins by western blotting. ANP was used as a positive control to assess cardiac hypertrophy in ISO infused mice. n = 4 mice per group. # marked western blotting images denotes SIRT2 antibody (#12650; Cell Signaling), used in this assay detects single band. ( F ) Histogram showing relative acetylated GSK3β in vehicle and ISO-treated mice heart tissues, as measured from . Signal intensities of acetylated GSK3β and GSK3β were measured by densitometry analysis (ImageJ software). n = 4 mice per group. Data is presented as mean ± s.d. *p<0.05. Student’s t test was used to calculate the p values. ( G ) GSK3β was immunoprecipitated from heart tissues of 8 weeks old 129/Sv mice using anti-GSK3β antibody (sc-9166, Santa Cruz Biotechnology), and the affinity resin with protein A/G immobilized. Western blotting was performed to detect GSK3β interaction with p300 using anti-p300 antibody. IgG was used as a negative control. Whole cell lysates (WCL) were probed for the presence of GSK3β and p300 by western blotting. ( H ) Co-localization of GSK3β with p300 was assessed in 293 T cells by confocal microscopy. The antibodies used are anti-GSK3β (sc-9166, Santacruz), and p300 (05–257, Millipore). DAPI was used to stain the nucleus. Expanded images (right small boxes) show yellow color in the merge image, indicating the co-localization of GSK3β (Green) and p300 (Red) in the nucleus. ( I ) In vitro binding assay to test the direct interaction between GSK3β and p300. Recombinant p300 (Millipore # 2273152) was incubated with recombinant GST or GST-GSK3β, purified from E. coli BL21 (DE3) by affinity chromatography using Glutathione Sepharose 4B. ( J ) Western blotting analysis showing the acetylation and activity of GSK3β in rat neonatal cardiomyocytes infected with adenovirus expressing either luciferase shRNA (control) or p300 shRNA (p300-KD) for 72 hr. Depletion of p300 was confirmed by western blotting. GSK3β was immunoprecipitated from control and p300-KD cells using anti-GSK3β antibody (sc-9166, Santa Cruz Biotechnology) and the affinity resin immobilized with protein A/G. Western blotting was performed to detect acetylation of GSK3β using the anti Ac-Lysine antibody. GSK3β activity was measured by assessing the phosphorylation of glycogen synthase (p–GS). Site-specific antibodies were used to detect the phosphorylation of GSK3β at indicated residues in cardiomyocyte lysates (WCL). ( K ) Histogram showing the quantification of relative acetylated GSK3β in control and p300 depleted (p300-KD) rat neonatal cardiomyocytes, as measured from . Rat neonatal cardiomyocytes were infected with adenovirus expressing either luciferase shRNA (control) or p300 shRNA (p300-KD) for 72 hr. Signal intensities of acetylated GSK3β and GSK3β were quantified by densitometry analysis (ImageJ software). n = 3 independent experiments. Data is presented as mean ± s.d. *p<0.05. Student’s t test was used to calculate the p values. ( L ) Histogram depicting the activity of GSK3β in control and p300 depleted (p300-KD) rat neonatal cardiomyocytes, as measured by the ratio of phosphorylation of glycogen synthase vs total glycogen synthase from . Rat neonatal cardiomyocytes were infected with adenovirus expressing either luciferase shRNA (control) or p300 shRNA (p300-KD) for 72 hr. Signal intensities of phospho-glycogen synthase and glycogen synthase were measured by densitometry analysis (ImageJ software). n = 3 independent experiments. Data is presented as mean ± s.d. *p<0.05. Student’s t test was used to calculate the p values. ( M ) Western blotting analysis showing the acetylation of GSK3β in rat neonatal cardiomyocytes infected with either control (Ad-null) or p300 overexpressing adenovirus (Ad-p300) for 24 hr. Overexpression of p300 was confirmed by western blotting. GSK3β was immunoprecipitated using anti-GSK3β antibody (sc-9166, Santacruz) and the affinity resin with protein A/G immobilized. Site-specific antibodies were used to detect the phosphorylation of GSK3β at indicated residues in cell lysates (WCL). ( N ) Western blotting analysis showing the activity of GSK3β in rat neonatal cardiomyocytes infected with control (Ad-null) or p300 expressing adenovirus (Ad-p300) for 24 hr. Overexpression of p300 was confirmed by western blotting and the activity of GSK3β was probed by assessing the levels of p-GS and GS by western blotting. ( O ) Histogram showing the activity of GSK3β in control (Ad-Null) or p300 overexpressing (Ad-p300) rat neonatal cardiomyocytes, as measured by the ratio of phosphorylation of glycogen synthase vs total glycogen synthase from . Signal intensities of phospho-glycogen synthase and glycogen synthase were assessed by densitometry analysis (ImageJ software). n = 3 independent experiments. Data is presented as mean ± s.d. *p<0.05. Student’s t test was used to calculate the p values. ( P ) In vitro kinase assay showing the activity of acetylated and non-acetylated GSK3β. Human GSK3β with HA tag was overexpressed in HeLa cells by transfection of the plasmid pcDNA3-HA-GSK3β. HA-GSK3β was immunoprecipitated using HA-coupled agarose beads (Sigma-Aldrich) and the HA-GSK3β was acetylated by recombinant p300 (Millipore), in the presence or absence of Acetyl-CoA (Ac-CoA) in HAT buffer. The enzymatic activity of GSK3β was measured against glycogen synthase (GS)-peptide. n = 6 independent experiments. Data is presented as mean ± s.d. *p<0.05. One-way ANOVA was used to calculate the p values.

Article Snippet: Recombinant protein , p300 , Merck Millipore , 14–418 , http://dx.doi.org/10.1038/s41418-018-0069-8.

Techniques: Activity Assay, Immunoprecipitation, Incubation, Phospho-proteomics, Western Blot, Negative Control, Positive Control, Software, Confocal Microscopy, Staining, In Vitro, Binding Assay, Recombinant, Purification, Affinity Chromatography, Infection, Expressing, Luciferase, shRNA, Control, Over Expression, Kinase Assay, Transfection, Plasmid Preparation

Journal: eLife

Article Title: SIRT2 deacetylase regulates the activity of GSK3 isoforms independent of inhibitory phosphorylation

doi: 10.7554/eLife.32952

Figure Lengend Snippet: ( A ) Western blot analysis of acetylated GSK3β in heart samples of 9 months old WT and SIRT2-KO littermates. GSK3β was immunoprecipitated from heart tissue lysates of WT and SIRT2-KO mice using anti-GSK3β antibody (sc-9166, Santa Cruz Biotechnolgy), and the affinity resin immobilized with protein A/G. Western blotting was performed to detect GSK3β acetylation by anti-Ac-Lysine antibody. IgG was used as a negative control. Whole cell lysates (WCL) were probed for the SIRT2 and GAPDH by western blotting. n = 4 mice per group. ( B ) Histogram showing relative acetylated GSK3β in 9 months old WT and SIRT2-KO mice heart tissues, as measured from . Signal intensities of acetylated GSK3β and GSK3β were measured by densitometry analysis (ImageJ software). n = 4 mice per group. Data is presented as mean ± s.d, *p<0.05. Student’s t test was used to calculate the p values. ( C ) GSK3β was immunoprecipitated from heart tissue lysates of 8 weeks old 129/Sv mice using anti-GSK3β antibody (sc-9166, Santa Cruz Biotechnolgy ), and the affinity resin immobilized with protein A/G. GSK3β interaction with SIRT2 was tested by western blotting using anti-SIRT2 antibody. IgG was used as negative control. Heart lysates was probed for indicated proteins by western blotting. ( D ) In vitro binding assay to test the interaction between GSK3β and SIRT2. Flag-SIRT2 was overexpressed in 293 cells by a plasmid encoding human Flag-SIRT2. Recombinant His or His-GSK3β was purified from E. coli BL21 (DE3) by Ni-NTA affinity chromatography and were incubated with 293 T cell lysates overexpressing human Flag-SIRT2. Interaction between GSK3β and SIRT2 was tested by western blotting. # marked western images denotes SIRT2 antibody used in this assay detects single band. ( E ) In vitro deacetylation assay showing SIRT2 as GSK3β deacetylase. Human HA-GSK3β was overexpressed in HeLa cells by transfection of the plasmid pcDNA3-HA-GSK3β. HA-GSK3β was immunoprecipitated using HA-coupled agarose beads (Sigma-Aldrich) and the HA-GSK3β was acetylated by recombinant p300 (Millipore), in the presence or absence of Acetyl-CoA (Ac-CoA) in HAT buffer. The acetylated HA-GSK3β was further incubated with either Flag-tagged SIRT2 or SIRT2-H187Y, which were immunoprecipitated from HEK 293 cell lysates overexpressing respective plasmids encoding Flag-tagged WT or SIRT2-H187Y using agarose beads conjugated to anti-Flag antibody (Sigma A2220). The deacetylation reaction was carried out in the presence or absence of NAD + in a HDAC buffer. GSK3β acetylation was analyzed by western blotting using anti-Ac-Lysine antibody. # marked western images denotes SIRT2 antibody used in this assay detects single band. ( F ) In vitro kinase assay depicting the activity of acetylated and deacetylated GSK3β. Human HA-GSK3β was overexpressed in HeLa cells by transfection of the plasmid pcDNA3-HA-GSK3β. Recombinant HA-GSK3β was immunoprecipitated using HA-coupled beads and was acetylated by recombinant p300 in the presence or absence of Acetyl-CoA (Ac-CoA) in HAT buffer. Acetylated GSK3β was further deacetylated by either Flag-tagged WT or SIRT2-H187Y (SIRT2-HY), a catalytic inactive mutant of SIRT2, which was immunoprecipitated from HEK 293 cells, overexpressed with plasmid encoding Flag-tagged WT or SIRT2-H187Y using agarose beads conjugated to anti-Flag antibody (Sigma A2220). The deacetylation reaction was carried out in the presence or absence of NAD + in a HDAC buffer and further enzymatic activity of GSK3β was measured against glycogen synthase (GS)-peptide, as described in the Materials and methods section. n = 5. Data is presented as mean ± s.d. *p<0.05. One-way ANOVA was used to calculate the p values. ( G ) Western blot analysis of acetylated GSK3β from control or SIRT2-depleted (SIRT2-KD) cardiomyocytes. Neonatal rat cardiomyocytes were transfected with either non-targeting (control) or siRNA targeting SIRT2 using Lipofectamine RNAiMAX reagent for 72 hr. SIRT2 depletion was confirmed by Western blotting. Total cellular acetylation was probed by anti-Ac-Lysine antibody to test the effect of SIRT2 depletion in cardiomyocytes. GSK3β was immunoprecipitated from these cell lysates using anti-GSK3β antibody (sc-9166, Santa Cruz Biotechnolgy), and the affinity resin immobilized with protein A/G. Western blotting was performed to detect acetylation of GSK3β by anti-Ac-Lysine antibody. Cell lysates (WCL) from control and SIRT2-KD cardiomyocytes were probed for indicated proteins by western blotting. ( H ) Western blotting analysis of hearts lysates from 9 months old WT and SIRT2-KO mice littermates for indicated proteins. n = 4 mice per group. ( I ) Histogram showing activity of GSK3β in WT and SIRT2-KO mice hearts at 9 months of age. GSK3β was immunoprecipitated from the heart lysates of WT and SIRT2-KO mice using anti-GSK3β antibody, clone GSK-4B (Sigma). The immunoprecipitated GSK3β was incubated with the peptide substrate in the presence of γ− 32 P-ATP. The incorporation of 32 P into the GSK3β Peptide Substrate, which contains specific phosphorylation residue of GSK3β was measured. n = 6 mice per group. Data is presented as mean ± s.d. *p<0.05. Student’s t test was used to calculate the p values. ( J ) In vitro deacetylation assay to test whether SIRT2 deacetylates K183 residue of GSK3β. HA-tagged GSK3β or GSK3β-K183R was overexpressed in HeLa cells and was immunoprecipitated using HA-coupled beads. HA-tagged WT-GSK3β or GSK3β-K183R were incubated with Flag-SIRT2 immunoprecipitated from HEK 293 T cells using agarose beads conjugated to Anti-Flag antibody (Sigma A2220). The deacetylation reaction was carried out in the presence or absence of NAD + in a deacetylation buffer. Acetylation status of GSK3β was analyzed by western blotting. # marked western images denotes SIRT2 antibody used in this assay detects single band. ( K ) Histogram showing relative acetylation of HA-tagged GSK3β or GSK3β-K183R, which was incubated with Flag-SIRT2. The data is generated from . Signal intensities of acetylated-GSK3β and GSK3β were measured by densitometry analysis (ImageJ software). n = 4 independent experiments. Data is presented as mean ± s.d. *p<0.05. One-way ANOVA was used to calculate the p values. ( L ) Histogram showing binding of γ− 32 P-ATP to acetylated and deacetylated His-GSK3β. Recombinant His-GSK3β was purified from E. coli BL 21 (DE3) by Ni-NTA affinity chromatography. Purified His-GSK3β was acetylated by recombinant p300 in the presence of Ac-CoA in HAT buffer. Acetylated His-GSK3β was further deacetylated by Flag-SIRT2 immunoprecipitated from HEK 293 T cells. The binding of γ− 32 P-ATP to acetylated and deacetylated His-GSK3β was assessed by the protocol described in Materials and methods section. n = 4. Data is presented as mean ± s.d. *p<0.05. One-way ANOVA was used to calculate the p values. ( M ) Histogram showing activity of WT or mutants of GSK3β. HA-tagged WT or mutants of GSK3β was immunoprecipitated from HeLa cells transfected with respective plasmids using HA-coupled agarose beads. The enzymatic activity of GSK3β was measured against glycogen synthase (GS)-peptide, as described in the Materials and methods section. n = 4. Data is presented as mean ± s.d. *p<0.05. One-way ANOVA was used to calculate the p values.

Article Snippet: Recombinant protein , p300 , Merck Millipore , 14–418 , http://dx.doi.org/10.1038/s41418-018-0069-8.

Techniques: Western Blot, Immunoprecipitation, Negative Control, Software, In Vitro, Binding Assay, Plasmid Preparation, Recombinant, Purification, Affinity Chromatography, Incubation, Histone Deacetylase Assay, Transfection, Kinase Assay, Activity Assay, Mutagenesis, Control, Phospho-proteomics, Residue, Generated

Journal: eLife

Article Title: SIRT2 deacetylase regulates the activity of GSK3 isoforms independent of inhibitory phosphorylation

doi: 10.7554/eLife.32952

Figure Lengend Snippet: ( A ) Western blot analysis showing acetylation status of both isoforms of GSK3 in control or SIRT2 depleted (SIRT2-KD) cardiomyocytes. Neonatal rat cardiomyocytes were transfected with either non-targeting or siRNA pool targeting SIRT2 using Lipofectamine RNAiMAX reagent for 72 hr. SIRT2 depletion was confirmed by western blotting. GSK3 was immunoprecipitated from cell lysates using anti-GSK3 antibody and the affinity resin immobilized with protein A/G. Western blotting was performed to detect GSK3α/β acetylation by anti-Ac-Lysine antibody. Cell lysates was probed for SIRT2 and actin antibodies by western blotting. ( B ) Histogram showing relative acetylated-GSK3α and GSK3β in control and SIRT2-depleted (SIRT2-KD) cardiomyocytes, as measured from . Signal intensities of acetylated-GSK3α and acetylated-GSK3β were measured by densitometry analysis (ImageJ software). n = 3. Data is presented as mean ± s.d. *p<0.05. Student’s t test was used to calculate the p values. ( C ) Histogram showing enzymatic activity of acetylated and deacetylated GSK3α. Recombinant HA- GSK3α was immunoprecipitated from HeLa cells overexpressing pcDNA-HA-GSK3α using HA-coupled agarose beads. Immunoprecipitated HA-GSK3α was acetylated by p300 in the presence of Acetyl-CoA (Ac-CoA) in HAT buffer. Acetylated GSK3α was further deacetylated by Flag-SIRT2 immunoprecipitated from HEK 293 T cells overexpressing plasmid encoding Flag-tagged SIRT2-WT using agarose beads conjugated to anti-Flag antibody (Sigma A2220). The enzymatic activity of GSK3α was measured against glycogen synthase (GS)-peptide. n = 5. Data is presented as mean ± s.d. *p<0.05. One-way ANOVA was used to calculate the p values. ( D ) Annotation of representative tandem mass spectra of trypsin-digested GSK3α, depicting K99, K246 acetylation. ( E ) Protein sequence alignment of the modeled region of GSK3α and the structure of GSK3β. ( F ) Cartoon, surface representation of the homology model of GSK3α (highlighted is the adenine nucleotide-binding pocket and position of K246 residue).

Article Snippet: Recombinant protein , p300 , Merck Millipore , 14–418 , http://dx.doi.org/10.1038/s41418-018-0069-8.

Techniques: Western Blot, Control, Transfection, Immunoprecipitation, Software, Activity Assay, Recombinant, Plasmid Preparation, Sequencing, Binding Assay, Residue

Journal: eLife

Article Title: SIRT2 deacetylase regulates the activity of GSK3 isoforms independent of inhibitory phosphorylation

doi: 10.7554/eLife.32952

Figure Lengend Snippet: ( A ) Western blotting analysis depicting the activity of GSK3 inhibitor X (GSK3-X). Neonatal rat cardiomyocytes were treated with vehicle or 500 nM GSK3-X for 48 hr and the activity of GSK3 was assessed by monitoring the phosphorylation of GS by specific antibody. ( B ) [ 3 H]-leucine incorporation into total cellular protein of control (Ad-GFP) or SIRT2-overexpressing (Ad-SIRT2) rat neonatal cardiomyocytes treated with either vehicle or 500 nM GSK3 inhibitor X (GSK3-X) for 48 hr. Cardiomyocytes were infected with adenoviral vectors encoding either GFP or SIRT2 for 24 hr prior to GSK3-X treatment. After the GSK3-X treatment, cardiomyocytes were stimulated with either vehicle or 20 µM ISO for 24 hr and the [ 3 H]-leucine incorporation was monitored. c.p.m. counts per minute. n = 10. Data is presented as mean ± s.d. *p<0.05. Two-way ANOVA was used to calculate the p values. ( C ) Histogram showing quantification of relative cardiomyocyte area in control (Ad-Null) and SIRT2-overexpressing (Ad-SIRT2) rat neonatal cardiomyocytes treated with either vehicle or 500 nM GSK3 inhibitor X (GSK3-X) for 48 hr. Cardiomyocytes were infected with adenoviral vectors encoding either control or SIRT2 for 24 hr prior to GSK3-X treatment. After the GSK3-X treatment, cardiomyocytes were stimulated with either vehicle or 20 µM ISO for 24 hr and the relative cardiomyocyte area is quantified as described in Materials and methods section. Data is presented as mean ± s.d. *p<0.05. Two-way ANOVA was used to calculate the p values. ( D ) Representative confocal images depicting perinuclear expression of ANP in control (Ad-Null) or SIRT2-overexpressing (Ad-SIRT2) cardiomyocytes treated with either vehicle or ISO (20 µM, 24 hr), with or without GSK3 inhibitor X (GSK3-X, 500 nM, 48 hr). Scale bar = 20 µm. ANP (Green), Myomesin (Red), Hoechst (Blue). ( E ) Western blotting analysis for puromycin incorporation in control or SIRT2-depleted (SIRT2-KD) neonatal rat cardiomyocytes infected with adenovirus expressing either control (Ad-luc shRNA) or p300 shRNA (Ad-p300-shRNA) 48 hr. p300 depletion was confirmed by western blotting. Pulse of puromycin was given 30 min prior to harvesting of cardiomyocytes and puromycin incorporation into nascent proteins was tested using anti-puromycin antibody. # marked Western images denotes SIRT2 antibody used in this assay detects single band. ( F ) Histogram showing relative puromycin levels in control or SIRT2-depleted (SIRT2-KD) cardiomyocytes infected with adenovirus expressing either control (Ad-luc shRNA) or p300 shRNA (Ad-p300-shRNA). The data is generated from . Signal intensities of puromycin and actin were measured by densitometry analysis using ImageJ software. n = 3 independent experiments. Data is presented as mean ± s.d. *p<0.05. Two-way ANOVA was used to calculate the p values. ( G ) Western blotting analysis of GSK3β acetylation and activity in heart lysates of vehicle or anacardic acid (p300 inhibitor) treated 9 months old WT and SIRT2-KO mice littermates. Anacardic acid was injected intraperitoneal at the dose of 5 mg/kg/day for 10 days in mice. Peanut oil was used as vehicle. GSK3β was immunoprecipitated from heart lysates of WT and SIRT2-KO mice using anti-GSK3β antibody (sc-9166, Santa Cruz Biotechnology), and the affinity resin with protein A/G immobilized. Western blotting was performed to detect GSK3β acetylation by anti-Ac-Lysine antibody. GSK3β activity was measured by detecting the phosphorylation of GS. SIRT2 depletion was confirmed by western blotting. Whole cell lysates (WCL) was probed for indicated proteins by western blotting. ( H ) Histogram showing relative GSK3β acetylation in heart lysates of vehicle or anacardic acid (5 mg/kg/day for 10 days) treated 9 months old WT and SIRT2-KO mice from . n = 3. Signal intensities of GSK3β and acetylated-GSK3β was measured by densitometry analysis using ImageJ software. Data is presented as mean ± s.d. *p<0.05. Two-way ANOVA was used to calculate the p values. ( I ) Scatter plot depicting HW/TL ratio of 9 months old WT and SIRT2-KO mice treated with either vehicle or anacardic acid, (p300-INH), at the dose of 5 mg/kg/day for 10 days. n = 5 mice per group. Data is presented as mean ± s.d. *p<0.05. Two-way ANOVA was used to calculate the p values. ( J ) Scatter plot showing left ventricular posterior wall thickness of 9 months old WT and SIRT2-KO mice treated with either vehicle or anacardic acid (p300-INH), at the dose of 5 mg/kg/day for 10 days. n = 6–8 mice per group. Data is presented as mean ± s.d. *p<0.05. Two-way ANOVA was used to calculate the p values. ( K ) Scatter plot depicting cardiac contractile functions, as measured by ejection fraction of 9 months old WT and SIRT2-KO mice treated with either vehicle or anacardic acid (p300-INH), at the dose of 5 mg/kg/day for 10 days. n = 6–8 mice per group. Data is presented as mean ± s.d. *p<0.05. Two-way ANOVA was used to calculate the p values.

Article Snippet: Recombinant protein , p300 , Merck Millipore , 14–418 , http://dx.doi.org/10.1038/s41418-018-0069-8.

Techniques: Western Blot, Activity Assay, Phospho-proteomics, Control, Infection, Expressing, shRNA, Generated, Software, Injection, Immunoprecipitation

Journal: eLife

Article Title: SIRT2 deacetylase regulates the activity of GSK3 isoforms independent of inhibitory phosphorylation

doi: 10.7554/eLife.32952

Figure Lengend Snippet:

Article Snippet: Recombinant protein , p300 , Merck Millipore , 14–418 , http://dx.doi.org/10.1038/s41418-018-0069-8.

Techniques: Knock-Out, Western Blot, Agarose Gel Electrophoresis, Produced, Transfection, Construct, Plasmid Preparation, Modification, Infection, Recombinant, Sequencing, Activity Assay, Mutagenesis, Protease Inhibitor, Microscopy, Software, Membrane, Cell Culture

Journal: bioRxiv

Article Title: MECOM is a master repressor of myeloid differentiation through dose control of CEBPA in acute myeloid leukemia

doi: 10.1101/2025.01.02.631046

Figure Lengend Snippet: . Volcano plot of differential chromatin accessibility (ATAC-seq) of untreated (n = 3) versus auxin-treated for 4 hrs (n = 2; 500 µM) calculated with DiffBind. . Volcano plot of differential chromatin accessibility (ATAC-seq) of untreated (n = 3) versus auxin-treated for 24 hrs (n = 2; 500 µM) calculated with DiffBind. All labels containing “CEBPA” at FDR < 0.05 are labelled in bold. . ATAC-seq and ChIP-seq for indicated factors in unmodified cells (MECOM tracks; inv(3) patient and MUTZ3 bulk) and a MUTZ3 MECOM-V5-AID OsTIR1 clone (ATAC, RUNX1, CTBP2, P300, H3K27ac), untreated and auxin-treated for indicated timepoints (500 µM). Coordinates are hg19 and tracks are RPKM normalized.

Article Snippet: The following antibodies were used in ChIP-seq: H3K27AC (Diagenode cat C15410196, lot A1723-0041D), RUNX1 (Abcam cat AB23980, lot GR3364239-1), P300 (Diagenode cat C15200211, lot 001), H3K9ME3 (Diagenode, cat no C15410193, lot A2217P), H3K27ME3 (Diagenode cat no C15410195, lot A0824DD), CTBP2 (BD cat no 612044, lot7069947) and V5 (ThermoFisher #R960-25).

Techniques: ChIP-sequencing

Journal: bioRxiv

Article Title: MECOM is a master repressor of myeloid differentiation through dose control of CEBPA in acute myeloid leukemia

doi: 10.1101/2025.01.02.631046

Figure Lengend Snippet: . Volcano plot of ATAC-seq footprinting of on MUTZ3-V5-AID-OsTIR1 cells after 4 hrs of auxin depletion (n = 2) compared to untreated control (n = 4), as calculated with TOBIAS. Each dot is a single motif, and motifs are colored by their cluster. Composite motifs of the indicated motif clusters are shown in the plot. Composite GATA motif for panel is shown on the right-hand side. . Volcano plot differential P300 binding (ChIP-seq) in MUTZ3-V5-AID-OsTIR1 cells after 24 hrs of auxin depletion versus untreated control (n = 2). Details of peak annotation in the Methods. AME motif enrichment is shown on the corresponding side of the plot. Bars represent the fraction of peaks that contain a high-score motif per set; significance is indicated with AME e-value across the bars. Top-8 motifs are shown per ChIP at an e-value cut-off is 0.05. All labels containing “CEBPA” at FDR < 0.05 are labelled in bold. . Volcano plot of differential RUNX1 binding (ChIP-seq) in MUTZ3-V5-AID-OsTIR1 cells after 24 hrs of auxin depletion versus untreated control (n = 2). Peak annotation is described in the methods. AME motif enrichment is shown on the corresponding side of the plot. Bars represent the fraction of peaks that contain a high-score motif per set; significance is indicated with AME e-value across the bars. Top-8 motifs are shown per ChIP at an e-value cut-off is 0.05. All labels containing “CEBPA” at FDR < 0.05 are labelled in bold.

Article Snippet: The following antibodies were used in ChIP-seq: H3K27AC (Diagenode cat C15410196, lot A1723-0041D), RUNX1 (Abcam cat AB23980, lot GR3364239-1), P300 (Diagenode cat C15200211, lot 001), H3K9ME3 (Diagenode, cat no C15410193, lot A2217P), H3K27ME3 (Diagenode cat no C15410195, lot A0824DD), CTBP2 (BD cat no 612044, lot7069947) and V5 (ThermoFisher #R960-25).

Techniques: Footprinting, Control, Binding Assay, ChIP-sequencing

Journal: Neurotherapeutics

Article Title: SIRT2 Inhibition Improves Functional Motor Recovery After Peripheral Nerve Injury

doi: 10.1007/s13311-020-00860-3

Figure Lengend Snippet: (A) Top: Confocal images of motoneurons (MNs) immunolabeled with antibodies against Ac-H3K9, Ac-p53 (k373), Ac-p300 (k1542), and p300 in red and counterstained with FluoroNissl (green) and DAPI (blue) from MNs of wild-type (WT) and SIRT2 KO animals at 30 dpi. Scale bar = 100 μm and 20 μm. Down: Bar graphs of the mean of the Ac-H3k9, Ac-p53, and the ratio of Ac-p300/total p300 immunofluorescence intensity inside the cytoplasm or nuclei from injured MNs (n = 4, t test, *P < 0.05 vs. WT). (B) Representative microphotographs and bar graphs showing the mean (± SEM) values of neurite elongation and maximum neurite length per cell from SH-SY5Y cells after 24 h of treatment with vehicle (Veh), DMOG, AK7, C646, or AK7+C646, or eGFP, eGFP+AK7, shRNA/p300, and shRNA/p300+AK7. Insets are a black and white magnification of a single cell at each condition (n = 3–4, ANOVA, post hoc Bonferroni *P < 0.05 vs. Veh. Scale bar = 100 μm; inset, 20 = μm). (C) Western blots and histogram showing the analysis of p53, Ac-p53, and GAP43, in different experimental groups (n = 3–4, ANOVA, post hoc Bonferroni, *P < 0.05 vs. Veh, #P < 0.05 vs. AK7)

Article Snippet: Slices were incubated overnight at 4 °C with the following primary antibodies: rabbit-anti NAD-dependent deacetylase sirtuin-2 (SIRT2; 1:200, Sigma), rabbit anti-acetyl-histone H3 (Lys9) (Acetyl H3-K9; 1:50, Millipore), rabbit anti-acetyl-p53 (Lys373) (Acetyl p53-K373; 1:500, Millipore), rabbit anti-phosphoprotein kinase B/AKT (Ser-473) (pAKT; 1:500, Santa Cruz), rabbit anti-growth-associated protein 43 (GAP43) (1:1000; Millipore), rabbit anti-acetyl-p300 (Lys 1542) (1:200; Bionova), and mouse anti-p300 (1:200; Bionova).

Techniques: Immunolabeling, Immunofluorescence, shRNA, Western Blot

Journal: Translational Andrology and Urology

Article Title: The effects of histone crotonylation and bromodomain protein 4 on prostate cancer cell lines

doi: 10.21037/tau-21-53

Figure Lengend Snippet: Primer sequence

Article Snippet: Anti-actin (β-actin) antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA), anti-histone H3 antibody and BRD4 antibody were purchased from Abcam (Cambridge, UK), and anti-histone acetylase P300 antibody was purchased from AbSci (Vancouver, WA, USA).

Techniques: Sequencing