Journal: Molecular Biology of the Cell

Article Title: Grainyhead-like 2 inhibits the coactivator p300, suppressing tubulogenesis and the epithelial–mesenchymal transition

doi: 10.1091/mbc.e16-04-0249

Figure Lengend Snippet: FIGURE 3: GRHL2 suppresses AP-1 and p300 function. (A) GRHL2 suppresses AP1 function. HT1080 and 293T cells were cotransfected with AP-1 response element-luciferase reporter construct and GRHL2 expression vector or empty vector. PMA was used to induce AP-1 signaling in 293T cells. Values represent relative luciferase activity normalized to TK-β- galactosidase control. (B) Venn diagram comparing GRHL2-repressed genes (Farris et al., 2016) vs. p300 target genes identified via E1a (Ferrari et al., 2014). (C) GRHL2 suppresses the p300 pathway. p300 effector and target genes induced by HGF in MDCK cells with GRHL2 shRNA vs. cells with constitutive GRHL2 determined by Ingenuity Pathway Analysis. Quantitation of the number of unregulated, HGF up-regulated, or HGF down-regulated genes for both cell lines. The p300 interactome diagram on which this is based is shown in Supplemental Figure S7. (D) GRHL2 suppresses p300 function. GAL4-minimal promoter-luciferase activation by a cotransfected GAL4-p300 was assayed in HT1080 cells in the presence or absence of cotransfected GRHL2 expression vector; values represent relative luciferase activity normalized to TK-β-galactosidase control.

Article Snippet: After lysates were precleared at 13,200 rpm, 10 μg of p300 antibody (SCBT, SC-584) or rabbit immunoglobulin G (IgG; Jackson, Bar Harbor, ME) was added to lysates and incubated overnight on a 4°C wheel.

Techniques: Luciferase, Construct, Expressing, Plasmid Preparation, Activity Assay, Control, shRNA, Quantitation Assay, Activation Assay

Journal: Molecular Biology of the Cell

Article Title: Grainyhead-like 2 inhibits the coactivator p300, suppressing tubulogenesis and the epithelial–mesenchymal transition

doi: 10.1091/mbc.e16-04-0249

Figure Lengend Snippet: FIGURE 4: GRHL2 suppresses the HAT activity of p300. (A) In vitro HAT assays with recombinant H3, p300, and GRHL2 proteins. Coomassie stains to assess the quality of recombinant proteins used throughout this study are shown in Supplemental Figure S19. (B) GRHL2 inhibits the acetylation of H3 on GRHL2-repressed but not GRHL2-induced promoters in vivo (ChIP assay). Cross-linked chromatin from HT1080+ vector HT-1080+GRHL2 cells immunoprecipitated with H3K27-Ac or rabbit IgG; the indicated promoters were assayed for H3K27Ac by qPCR using the primers in Materials and Methods. *p < 0.05. GRHL2 does not affect global histone H3K18-Ac or H3K27-Ac (Western blotting of total histones). (C) GRHL2 interacts with p300. Left, cotransfection of indicated expression constructs, followed by coimmunoprecipitation/Western blotting. Middle, coimmunoprecipitation of retrovirally expressed, S-tagged GRHL2 with endogenous p300. Right, coimmunoprecipitation of endogenous GRHL2 and endogenous P300.

Article Snippet: After lysates were precleared at 13,200 rpm, 10 μg of p300 antibody (SCBT, SC-584) or rabbit immunoglobulin G (IgG; Jackson, Bar Harbor, ME) was added to lysates and incubated overnight on a 4°C wheel.

Techniques: Activity Assay, In Vitro, Recombinant, In Vivo, Plasmid Preparation, Immunoprecipitation, Western Blot, Cotransfection, Expressing, Construct

Journal: Molecular Biology of the Cell

Article Title: Grainyhead-like 2 inhibits the coactivator p300, suppressing tubulogenesis and the epithelial–mesenchymal transition

doi: 10.1091/mbc.e16-04-0249

Figure Lengend Snippet: FIGURE 6: A small region within the DNA-binding domain of GRHL2, aa 425–437, inhibits p300. (A) Schematic of GRHL2 domains. DBD, DNA-binding domain; DD, dimerization domain; TAD, transactivation domain. (B) HAT assays using the indicated GRHL2 fragments, assayed as GST-fusion proteins. (C) GRHL2 aa 425–437 inhibits p300 HAT activity. The indicated fragments derived from GRHL2 were assayed as GST-fusions (left) or recombinant peptides (right) for inhibition of HAT activity. (D) GRHL2 aa 425–437 are required for inhibition of an AP-1 reporter, GAL4 reporter in conjunction with GAL4-p300, MMP1 reporter, or MMP14 reporter. Values represent relative luciferase activity normalized to TK-β-galactosidase control.

Article Snippet: After lysates were precleared at 13,200 rpm, 10 μg of p300 antibody (SCBT, SC-584) or rabbit immunoglobulin G (IgG; Jackson, Bar Harbor, ME) was added to lysates and incubated overnight on a 4°C wheel.

Techniques: Binding Assay, Activity Assay, Derivative Assay, Recombinant, Inhibition, Luciferase, Control

Journal: Molecular Biology of the Cell

Article Title: Grainyhead-like 2 inhibits the coactivator p300, suppressing tubulogenesis and the epithelial–mesenchymal transition

doi: 10.1091/mbc.e16-04-0249

Figure Lengend Snippet: FIGURE 7: The p300-inhibitory domain of GRHL2 is important for the suppression of tubulogenesis and reversion of EMT. (A) MDCK cells expressing GRHL2 wild-type or GRHL2 Δ425-437 were assayed for tubulogenesis (blue, nuclei; green, actin). Scale bar, 20 μm. Middle, quantitation; right, Western blot confirmation of protein levels. (B) MSP cells expressing GRHL2 wild-type or GRHL2 Δ425-437 were assayed for reversion of EMT by Western blotting for EMT markers (left), cell morphology (middle), or qPCR (right).

Article Snippet: After lysates were precleared at 13,200 rpm, 10 μg of p300 antibody (SCBT, SC-584) or rabbit immunoglobulin G (IgG; Jackson, Bar Harbor, ME) was added to lysates and incubated overnight on a 4°C wheel.

Techniques: Expressing, Quantitation Assay, Western Blot

Journal: Molecular Biology of the Cell

Article Title: Grainyhead-like 2 inhibits the coactivator p300, suppressing tubulogenesis and the epithelial–mesenchymal transition

doi: 10.1091/mbc.e16-04-0249

Figure Lengend Snippet: FIGURE 8: GRHL2 suppresses tubulogenesis and EMT by inhibition of p300.

Article Snippet: After lysates were precleared at 13,200 rpm, 10 μg of p300 antibody (SCBT, SC-584) or rabbit immunoglobulin G (IgG; Jackson, Bar Harbor, ME) was added to lysates and incubated overnight on a 4°C wheel.

Techniques: Inhibition

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: Scientific Reports

Article Title: Chromatin remodeling system p300-HDAC2-Sin3A is involved in Arginine Starvation-Induced HIF-1α Degradation at the ASS1 promoter for ASS1 Derepression

doi: 10.1038/s41598-017-11445-0

Figure Lengend Snippet: Effects of ADI on the regulation of HIF-1α stability by p300, HDAC2, and Sin3A. ( a ) ChIP assay of ASS1 promoter associations of p300, HDAC2 and Sin3A in A2058 cells treated with ADI for 15 min. ( b to d ) Effects of p300, Sin3A, and HDAC2 knockdown by siRNA as indicated on the expression of other proteins in the presence and absence of ADI (0.5 μg/ml, 1 hr). ( e to g ) ChIP assays of effects of p300, Sin3A, and HDAC2 knockdown on the ASS1 promoter associations of HIF1α, PHD2, HDAC2, p300, and Sin3A as indicated Cells were transfected with given siRNA as specified for 24 hr followed by ADI treatment (0.5 μg/ml) for 15 min.

Article Snippet: PCBP1(PA5–19409) antibody from Thermo Fisher Scientific; anti-LIMID1 antibody from Millipore Co.; rabbit anti-HDAC2, Sin3A, and p300 antibodies from Bethyl Laboratories; hydroxy-HIF-1α (Pro564) antibody and acetyl-Histone H3 Antibody Sampler Kit (#9927); antibodies of eukaryotic initiation factor 2α (eIF2α), phospho-eIF2α(S51), mammalian target of rapamycin (mTOR), phosphor-mTOR(S2448), p70S6K (T389), elf-4E-binding protein (4EBP1) and phosphor-4EBP1 (S65) were from Cell Signaling Technology; HDAC inhibitor (SAHA, suberoylanilide hydroxamic acid) was from Sigma-Aldrich; and HAT inhibitors PU139 was prepared as described previously , and I-CBP112 was obtained from Sigma-Aldridge.

Techniques: Knockdown, Expressing, Transfection

Journal: Scientific Reports

Article Title: Chromatin remodeling system p300-HDAC2-Sin3A is involved in Arginine Starvation-Induced HIF-1α Degradation at the ASS1 promoter for ASS1 Derepression

doi: 10.1038/s41598-017-11445-0

Figure Lengend Snippet: Effects of histone H3 deacetylation by ADI. ( a ) A2058 cells were treated with ADI for the time intervals as indicated, or with SAHA (20 μM for 1 hr as positive control), and lamin B expression (as control for sample loading). Acetylation status of various modified histones H3 were determined. ( b ) Reduction of ASS1-promoter associations of H3K27ac and H3K14ac by ADI (0.5 μg/ml, 15 min.). ( c to e ), Effects of p300, Sin3A, and HDAC2 knockdown by siRNAs, respectively, on the expression levels of H3K14ac and H3K27ac in A2058 cells treated with or without ADI. ( f ) Western blotting analyses of H3K14ac, H3K27ac, H3K9ac, and H3K18ac expression in A2058 and ADI-resistant (ADI R variants, 58R1 to 58R3). ( g ) ChIP assay of promoter-associations of p300, HDAC2, and HIF-1α in A2058 and ADI R cells (R1 to R3). ( h ) Western blotting analyses of HIF-1α, ASS1, H3K14ac, H3K27ac, and H3 expression in 4 matched pairs of primary cell lines derived from melanoma patients before (−) and after failed (+) by ADI treatments.

Article Snippet: PCBP1(PA5–19409) antibody from Thermo Fisher Scientific; anti-LIMID1 antibody from Millipore Co.; rabbit anti-HDAC2, Sin3A, and p300 antibodies from Bethyl Laboratories; hydroxy-HIF-1α (Pro564) antibody and acetyl-Histone H3 Antibody Sampler Kit (#9927); antibodies of eukaryotic initiation factor 2α (eIF2α), phospho-eIF2α(S51), mammalian target of rapamycin (mTOR), phosphor-mTOR(S2448), p70S6K (T389), elf-4E-binding protein (4EBP1) and phosphor-4EBP1 (S65) were from Cell Signaling Technology; HDAC inhibitor (SAHA, suberoylanilide hydroxamic acid) was from Sigma-Aldrich; and HAT inhibitors PU139 was prepared as described previously , and I-CBP112 was obtained from Sigma-Aldridge.

Techniques: Positive Control, Expressing, Control, Modification, Knockdown, Western Blot, Derivative Assay

Journal: Scientific Reports

Article Title: Chromatin remodeling system p300-HDAC2-Sin3A is involved in Arginine Starvation-Induced HIF-1α Degradation at the ASS1 promoter for ASS1 Derepression

doi: 10.1038/s41598-017-11445-0

Figure Lengend Snippet: Effects of ROS on ADI-induced HIF-1α degradation. ( a ) ADI-induced HF-1α degradation is inhibited by antioxidant, NAC. A2058 cells were treated with 10 μM MG-132 in the absence or presence of ADI or NAC (1 mM) for 4-h. Expression levels of HIF-1α, hydroxylated HIF-1α, and actin were determined by Western blotting. ( b ) The antioxidant NAC suppresses ADI-induced PHD2 enzymatic activity. A2058 cells were transfected with recombinant encoding HA-PHD2. Cells were treated with NAC or ADI as indicated for 1 hr. PHD2 enzymatic activity was measured using GST-ODDD (100 ng) as a substrate and production of HO-HIF-1α (p564). ( c ) Similar to those described in ( b ) was performed using anti-oxidants Mito-TEMPO (40 μM) and TEMPO (100 μM) for 1 hr. ( d , e ) Inhibitions of HDAC2 and PHD2 interaction by TEMPO in reciprocal co-IP assays. ( f , g ), Effects of antioxidants NAC (N, 1 mM) or TEMPO (T, 100 μM)) on ADI (A)-induced ASS1 promoter association of HIF-1 α , PHD2, p300, HDAC2 and H3K27ac in A2058 cells treated with or without ADI (A, 0.5 μg/ml, 1 hr) as determined by ChIP assay.

Article Snippet: PCBP1(PA5–19409) antibody from Thermo Fisher Scientific; anti-LIMID1 antibody from Millipore Co.; rabbit anti-HDAC2, Sin3A, and p300 antibodies from Bethyl Laboratories; hydroxy-HIF-1α (Pro564) antibody and acetyl-Histone H3 Antibody Sampler Kit (#9927); antibodies of eukaryotic initiation factor 2α (eIF2α), phospho-eIF2α(S51), mammalian target of rapamycin (mTOR), phosphor-mTOR(S2448), p70S6K (T389), elf-4E-binding protein (4EBP1) and phosphor-4EBP1 (S65) were from Cell Signaling Technology; HDAC inhibitor (SAHA, suberoylanilide hydroxamic acid) was from Sigma-Aldrich; and HAT inhibitors PU139 was prepared as described previously , and I-CBP112 was obtained from Sigma-Aldridge.

Techniques: Expressing, Western Blot, Activity Assay, Transfection, Recombinant, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Dosage amplification dictates oncogenic regulation by the NKX2-1 lineage factor in lung adenocarcinoma

doi: 10.1101/2023.10.26.563996

Figure Lengend Snippet: (A) ChIP-seq occupancy tracks for H3K27ac and H3K4me1 in 4 NKX2-1(+) LUAD cell lines. Union super-enhancer region ( NKX2-1 SE), with 10 candidate enhancers, is shown. (B) ChIP-seq occupancy tracks for H3K4me3, RNAPolII and p300, as well as ATAC-seq accessibility, in NCI-H2087 cells. (C) ChIP-seq occupancy tracks for NKX2-1, c-Jun, FOXA1/2, SOX2, and CTCF transcription factors in NCI-H2087 cells. (D) Immunoblot of NKX2-1 upon repression of the NKX2-1 SE, n=2 biological replicates. (E) Immunoblot of NKX2-1 upon activation of the NKX2-1 SE in NKX2-1(low) or NKX2-1(−) cells. (f-g). Luciferase enhancer activity for enhancers in the NKX2-1 super-enhancer using a (F) miniP or (G) NKX2-1 promoter reporter. (H) . Luciferase enhancer activity of a duplicated E7 enhancer shows >2x activity. (I-K) . Luciferase enhancer activity of E7 (I) region deletions, (J) minimal fragments, or (K) motif deletions. (F-K) Luciferase activity calculated relative to empty vector, error bars indicate mean±SEM, individual points labeled, n=3 biological replicates, two-tailed t-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Significance calculated against (G-H,J) empty vector control or (I,K) E7 full length. See also – .

Article Snippet: p300 (D2X6N) Rabbit mAb , CST , CAT#54062; RRID:AB_2799450.

Techniques: ChIP-sequencing, Western Blot, Activation Assay, Luciferase, Activity Assay, Plasmid Preparation, Labeling, Two Tailed Test, Control

Journal: bioRxiv

Article Title: Dosage amplification dictates oncogenic regulation by the NKX2-1 lineage factor in lung adenocarcinoma

doi: 10.1101/2023.10.26.563996

Figure Lengend Snippet: KEY RESOURCE TABLE

Article Snippet: p300 (D2X6N) Rabbit mAb , CST , CAT#54062; RRID:AB_2799450.

Techniques: Virus, Recombinant, Saline, Stripping Membranes, Transfection, Sterility, SYBR Green Assay, Staining, Ligation, Gel Extraction, Plasmid Preparation, Luciferase, Bicinchoninic Acid Protein Assay, Isolation, Mutagenesis, Sequencing, Expressing, CRISPR, Synthesized, shRNA, Software

Journal: Nature

Article Title: Transcription factor dimerization activates the p300 acetyltransferase

doi: 10.1038/s41586-018-0621-1

Figure Lengend Snippet: ( a ) Domain structure of p300. Sequence conservation of the AIL is shown using WebLogo . Constructs used are shown. ( b ) Analysis of in vitro expression of the indicated p300 variants. Purified proteins were analyzed for autoacetylation by immunoblotting with anti-p300 K1499 acetyl antibody (left panel), anti-FLAG antibody (middle) and Coomassie staining (right). Representative data of three independent experiments are shown. (c) Representative mass spec analysis of BRP_HAT_ZZ_ΔAIL following after in vitro expression (red curve) and after SIRT2 mediated deacetylation (black curve).

Article Snippet: Membranes were blocked with 5% skim milk in PBST buffer (PBS, 0.1% Tween-20) and probed with anti–p300 K1499ac rabbit polyclonal antibody (1:2,500 dilution; Cell Signaling, 4771), anti-Kac rabbit polyclonal antibody (1:2,500 dilution; Cell Signaling, 9441), anti-Flag mouse monoclonal antibody (1:2,500 dilution; Sigma, F1804), anti-HA rabbit polyclonal antibody (1:2500 dilution, Abcam, ab9110).

Techniques: Sequencing, Construct, In Vitro, Expressing, Purification, Western Blot, Staining, Mass Spectrometry

Journal: Nature

Article Title: Transcription factor dimerization activates the p300 acetyltransferase

doi: 10.1038/s41586-018-0621-1

Figure Lengend Snippet: (a) p300s was incubated for the indicated times in the presence or absence of inactive, monomeric IRF3 or TBK1-phosphorylated, dimeric pIRF3. Samples were analysed by SDS-PAGE followed by Coomassie staining and autoradiography. Representative data of three independent experiments are shown. ( b ) Quantification of autoacetylation of p300s. ( c ) p300 is activated by TBK1-mediated IRF3 phosphorylation. p300s was incubated with recombinant GST-STING, TBK1 and IRF3 in the presence of ATP and [ 14 C] acetyl-CoA. Top panel: Coomassie-stained SDS-PAGE gel. Middle panel: Analysis of IRF3 phosphorylation on S396 using immunoblotting. Bottom panel: autoradiography. Representative data of three independent experiments are shown. ( d ) HAT scintillation proximity assay. The degree of Histone H4 substrate acetylation was quantified using scintillation counting. ( e ) As in panel a but using inactive, monomeric STAT1ΔN or activated, dimeric pSTAT1ΔN. Activated, dimeric pSTAT1ΔNC lacking the C-terminal TAD did not stimulate p300s autoacetylation. Samples were analysed as in panel (a). Representative data of three independent experiments are shown. ( f ) Quantification of autoacetylation of p300s. Intensity values were normalized by dividing by the maximum autoacetylation signal obtained after 60 minutes. Error bars shown in panels ( b ), ( d ) and ( f ): Three independent experiments were performed and the mean value and error bars representing the standard deviation are shown. Data analysis and plotting was done with GraphPad Prism 7.0. For gel source data, see Supplementary Figure 1.

Article Snippet: Membranes were blocked with 5% skim milk in PBST buffer (PBS, 0.1% Tween-20) and probed with anti–p300 K1499ac rabbit polyclonal antibody (1:2,500 dilution; Cell Signaling, 4771), anti-Kac rabbit polyclonal antibody (1:2,500 dilution; Cell Signaling, 9441), anti-Flag mouse monoclonal antibody (1:2,500 dilution; Sigma, F1804), anti-HA rabbit polyclonal antibody (1:2500 dilution, Abcam, ab9110).

Techniques: Incubation, SDS Page, Staining, Autoradiography, Phospho-proteomics, Recombinant, Western Blot, Scintillation Proximity Assay, Standard Deviation

Journal: Nature

Article Title: Transcription factor dimerization activates the p300 acetyltransferase

doi: 10.1038/s41586-018-0621-1

Figure Lengend Snippet: ( a ) Superposition of the four p300 molecules (monomer I-IV) in the asymmetric crystallographic unit. While the Bromodomains (Bd), PHD and HAT domains superpose with a root mean square deviation of ~ 0.9 Å, the RING domains take multiple conformations, ( b ) 2Fo-Fc (blue mesh) as well as anomalous difference Fourier maps (orange mesh) for the four RING domains contoured around 1σ and 2.5σ, respectively.

Article Snippet: Membranes were blocked with 5% skim milk in PBST buffer (PBS, 0.1% Tween-20) and probed with anti–p300 K1499ac rabbit polyclonal antibody (1:2,500 dilution; Cell Signaling, 4771), anti-Kac rabbit polyclonal antibody (1:2,500 dilution; Cell Signaling, 9441), anti-Flag mouse monoclonal antibody (1:2,500 dilution; Sigma, F1804), anti-HA rabbit polyclonal antibody (1:2500 dilution, Abcam, ab9110).

Techniques:

Journal: Nature

Article Title: Transcription factor dimerization activates the p300 acetyltransferase

doi: 10.1038/s41586-018-0621-1

Figure Lengend Snippet: ( a ) There are four p300 molecules (monomer I-IV) in the asymmetric crystallographic unit. The four molecules show an antiparallel arrangement of the BRP-HAT domains. As a result HAT domains from monomer I and II are closely apposed. Monomer III and IV engage monomer IV sym and monomer III sym , respectively, of a neighboring crystallographic unit, showing that all promoters are in a AIL-loop swap conformation. Black arrows indicate the direction of the AIL. The disordered segment of the AIL is shown as a black dotted line. ( b ) Electron density of the AIL. A 2Fo-Fc and ( c ) a Fo-Fc difference density omit map contoured at 0.8 or 2.0 RMSD (root means square deviation), respectively. Coloring as in .

Article Snippet: Membranes were blocked with 5% skim milk in PBST buffer (PBS, 0.1% Tween-20) and probed with anti–p300 K1499ac rabbit polyclonal antibody (1:2,500 dilution; Cell Signaling, 4771), anti-Kac rabbit polyclonal antibody (1:2,500 dilution; Cell Signaling, 9441), anti-Flag mouse monoclonal antibody (1:2,500 dilution; Sigma, F1804), anti-HA rabbit polyclonal antibody (1:2500 dilution, Abcam, ab9110).

Techniques:

Journal: Nature

Article Title: Transcription factor dimerization activates the p300 acetyltransferase

doi: 10.1038/s41586-018-0621-1

Figure Lengend Snippet: ( a ) Simulations of the AIL in context of the loop-swapped dimer. Left panel: Cartoon of the trajectory of the AIL (dashed line). Right panel: Representative conformations with the AIL Cα backbone atoms are coloured according to charge. ( b ) SEC-MALLS analysis of deacetylated (blue) and acetylated (yellow) p300 core. Note the decrease in elution volume upon acetylation. ( c ) SEC-MALLS analysis of deacetylated (blue), acetylated (red) BRP_HAT_CH3 and deacetylated (black) and acetylated BRP_HAT_CH3 ΔAIL (green). There is no increase in elution volume upon acetylation of theΔAIL construct. ( d ) Comparison of acetylated and deacetylated BRP_HAT and BRP_HAT_CH3. The deacetylated BRP_HAT (green) and deacetylated BRP_HAT_CH3 (blue) elute at the same position indicating a similar hydrodynamic radius. The acetylated BRP_HAT (yellow) and BRP_HAT_CH3 (red) elute at a larger elution volume. The normalized refractive index is plotted as a function of elution volume from an S200 column coupled to a MALLS detector. Calculated molecular masses are plotted as a function of volume for each eluted peak. The experiment was carried out at least three times with similar results. One representative example of each sample is shown. ( e ) Mass spectrometry analysis using electrospray ionization (ESI) of the BRP_HAT before (blue), and after (yellow) autoacetylation. The molecular mass and the number of acetylation events are indicated. ( f ) BRP_HAT_CH3 before (blue) and after (red) autoacetylation. ( g ) BRP_HAT_CH3_ΔAIL before (black) and after (green) autoacetylation.

Article Snippet: Membranes were blocked with 5% skim milk in PBST buffer (PBS, 0.1% Tween-20) and probed with anti–p300 K1499ac rabbit polyclonal antibody (1:2,500 dilution; Cell Signaling, 4771), anti-Kac rabbit polyclonal antibody (1:2,500 dilution; Cell Signaling, 9441), anti-Flag mouse monoclonal antibody (1:2,500 dilution; Sigma, F1804), anti-HA rabbit polyclonal antibody (1:2500 dilution, Abcam, ab9110).

Techniques: Construct, Comparison, Refractive Index, Mass Spectrometry

Journal: Nature

Article Title: Transcription factor dimerization activates the p300 acetyltransferase

doi: 10.1038/s41586-018-0621-1

Figure Lengend Snippet: ( a ) The AIL contributes to histone substrate acetylation of activated p300. The details of the constructs used are indicated in . Defined amounts of p300 variants were incubated with acetyl-CoA and the indicated histones prior to SDS-PAGE analysis followed by Coomassie staining and western blotting with the indicated antibodies. ( b ) The indicated amounts of purified p300s variants were incubated with histone octamers as in panel ( a ), followed by SDS-PAGE and immunoblot analysis with the indicated antibodies. Anti-Kac: pan-acetyl-lysine antibody. Representative data of three independent experiments are shown. ( c ) Crystal structure of the H4 K12acK16ac peptide bound to the BΔRP module containing an in frame RING deletion. Amino acid residues 1169-1241 were replace by a single Glycine residue. The deletion removes the RING domain (black arrow) and does not adversely affect structural integrity of the BΔRP module. ( d ) Indicated variants of p300 were co-expressed with p53 in H1299 cells and analyzed by immunofluorescence with the indicated antibodies or ( e ) by western blotting. Representative data of three independent experiments are shown. Scale bar, 10 μm.

Article Snippet: Membranes were blocked with 5% skim milk in PBST buffer (PBS, 0.1% Tween-20) and probed with anti–p300 K1499ac rabbit polyclonal antibody (1:2,500 dilution; Cell Signaling, 4771), anti-Kac rabbit polyclonal antibody (1:2,500 dilution; Cell Signaling, 9441), anti-Flag mouse monoclonal antibody (1:2,500 dilution; Sigma, F1804), anti-HA rabbit polyclonal antibody (1:2500 dilution, Abcam, ab9110).

Techniques: Construct, Incubation, SDS Page, Staining, Western Blot, Purification, Residue, Immunofluorescence

Journal: Nature

Article Title: Transcription factor dimerization activates the p300 acetyltransferase

doi: 10.1038/s41586-018-0621-1

Figure Lengend Snippet: ( a ) Indicated variants of p300 were transiently co-transfected with p53 in COS cells and samples analysed by western blotting using the indicated antibodies. Bottom panel: quantification p300 K1499Ac signal. ( b ) Analysis of p53 acetylation. Bottom panel: quantification p53 acetylation signal. Representative data of three independent experiments are shown. For details on the mutants see . Arg and Glu: lysine amino acids in the AIL segment spanning amino acids 1546-1570 were mutated to arginine or glutamate, respectively ( c ) H1299 cells were transfected with the indicated construct and analyzed by immunoflorescence using Anti-HA for p300 (green) and cell nuclei were stained with Hoechst (blue). Bottom panels: Cells were treated with the A-485 HAT or the CBP30 Bromodomain inhibitor. Percentage of cells showing the indicated phenotype (n=200 cells) is indicated below each panel. Scale bar, 10 μm. For gel source data, see Supplementary Figure 1.

Article Snippet: Membranes were blocked with 5% skim milk in PBST buffer (PBS, 0.1% Tween-20) and probed with anti–p300 K1499ac rabbit polyclonal antibody (1:2,500 dilution; Cell Signaling, 4771), anti-Kac rabbit polyclonal antibody (1:2,500 dilution; Cell Signaling, 9441), anti-Flag mouse monoclonal antibody (1:2,500 dilution; Sigma, F1804), anti-HA rabbit polyclonal antibody (1:2500 dilution, Abcam, ab9110).

Techniques: Transfection, Western Blot, Construct, Staining

Journal: Nature

Article Title: Transcription factor dimerization activates the p300 acetyltransferase

doi: 10.1038/s41586-018-0621-1

Figure Lengend Snippet: Thermodynamic analysis of the interaction between p300 BRP and histone peptides by ITC. Mean and s.d. were determined from experiments performed in triplicate. Horizontal lines separate experiments involving different histone peptides or different protein constructs. Histone peptide sequences: H3 (1-20) ARTKQTQRKSTGGKAPRKQL, H3 (11-30) TGGKAPRKQLATKASRKSAP, H4 (4-24) GKGGKGLGKGGAKRHRKVLRD. AIL (Autoinhibitory loop peptide) SKNAKKKNNKKTSKNKSS (1545-1562). No binding was detected to non-acetylated peptides, or with a construct containing a mutation that abolishes acetyllysine binding (N1132A). *binding stoichiometry

Article Snippet: Membranes were blocked with 5% skim milk in PBST buffer (PBS, 0.1% Tween-20) and probed with anti–p300 K1499ac rabbit polyclonal antibody (1:2,500 dilution; Cell Signaling, 4771), anti-Kac rabbit polyclonal antibody (1:2,500 dilution; Cell Signaling, 9441), anti-Flag mouse monoclonal antibody (1:2,500 dilution; Sigma, F1804), anti-HA rabbit polyclonal antibody (1:2500 dilution, Abcam, ab9110).

Techniques: Construct, Binding Assay, Mutagenesis

Journal: Nature

Article Title: Transcription factor dimerization activates the p300 acetyltransferase

doi: 10.1038/s41586-018-0621-1

Figure Lengend Snippet: Summary of SEC-MALLS and mass spectrometry experiments. Column labelling: Molar masses determined by Mass spectrometry ( MM MS ), MM th the theoretical molar mass calculated from the appropriate primary sequences. Acetylation levels were estimated based on the mass differences as compared to the non-acetylated sample. MM SLS (Molar masses determined by SEC-MALLS) at a concentration of 2 mg·ml -1 . All p300 constructs contained the mutation Y1467F. The experiment was carried out at least three times with consistency. Results from one representative example are shown. The mass and errors reported for SEC-MALLS are the weight average molar mass and residual standard deviations of the observed data from the fitted values calculated using ASTRA.

Article Snippet: Membranes were blocked with 5% skim milk in PBST buffer (PBS, 0.1% Tween-20) and probed with anti–p300 K1499ac rabbit polyclonal antibody (1:2,500 dilution; Cell Signaling, 4771), anti-Kac rabbit polyclonal antibody (1:2,500 dilution; Cell Signaling, 9441), anti-Flag mouse monoclonal antibody (1:2,500 dilution; Sigma, F1804), anti-HA rabbit polyclonal antibody (1:2500 dilution, Abcam, ab9110).

Techniques: Mass Spectrometry, Concentration Assay, Construct, Mutagenesis

Journal: Nature

Article Title: Transcription factor dimerization activates the p300 acetyltransferase

doi: 10.1038/s41586-018-0621-1

Figure Lengend Snippet: (a) p300 is maintained in the inactive state by deacetylases such as SIRT2. IRF3 is autoinhibited by a C-terminal segment in the IAD domain. (b) TBK1 phosphorylation activates and dimerises IRF3. The activated IRF3 dimer engages the IBID domain of p300. (c) Recruitment of two molecules of p300 results in trans-autoacetylation in the AIL loop and HAT activation. (d ) Activated p300 can acetylate chromatin and engage acetylated substrates via the Bd. ( e ) A constant amount of p300s (2 μM) was incubated in [ 14 C] acetyl-CoA alone or in the presence of 2 μM Klf6 eRNA for the indicated time points. Samples were analyzed by SDS-PAGE followed by Coomassie staining (top) and autoradiography (bottom). ( f ) As in ( e ) but in the presence of 0.5 mM EDTA. The experiment was carried out at least two times with consistency. One representative example is shown. ( g ) Quality control of Klf6 RNA. 3 μg Klf6 was deposited on a 1% Agarose gel or a 14% 6M Urea PAGE gel and detected by SYBR Safe stain. M: 100bp DNA ladder (NEB).

Article Snippet: Membranes were blocked with 5% skim milk in PBST buffer (PBS, 0.1% Tween-20) and probed with anti–p300 K1499ac rabbit polyclonal antibody (1:2,500 dilution; Cell Signaling, 4771), anti-Kac rabbit polyclonal antibody (1:2,500 dilution; Cell Signaling, 9441), anti-Flag mouse monoclonal antibody (1:2,500 dilution; Sigma, F1804), anti-HA rabbit polyclonal antibody (1:2500 dilution, Abcam, ab9110).

Techniques: Phospho-proteomics, Activation Assay, Incubation, SDS Page, Staining, Autoradiography, Control, Agarose Gel Electrophoresis

Journal: Molecular and Cellular Biology

Article Title: The Modified Human DNA Repair Enzyme O 6 -Methylguanine-DNA Methyltransferase Is a Negative Regulator of Estrogen Receptor-Mediated Transcription upon Alkylation DNA Damage

doi: 10.1128/mcb.21.20.7105-7114.2001

Figure Lengend Snippet: FIG. 4. R-MGMT disrupts the ER–SRC-1 interaction and fails to associate with the CBP/p300 complex. (A) Quantification of MGMT and ER proteins. Lanes labeled MCF7 and T47D contain 200 g of total cell extracts, whereas lanes with numbers contain the purified recombinant MGMT and ER proteins, with numbers indicating amounts in nanograms (ng). Western blotting was performed with Mab.3B8 for MGMT and with Pab.HC20 for ER. (B) Immunoblot analysis of SRC-1 and ER in SRC-1 immunoprecipitates from MCF7 nuclear extracts. The lanes labeled Ctr, Mel, and 6BG are untreated (control), Mel (1 mM)-treated, and 6BG (50 M)-treated cells grown in full medium for 2 h, respectively. The top panel is the immunoblot of the SRC-1 protein visualized by Pab.N19 in the SRC-1 Mab.677-F11 immunoprecipitate, and the bottom panel is the coimmunoprecipitated ER shown by Pab.HC20. (C) Immunoblot analysis of ER (Mab.F10) and MGMT or R-MGMT (Mab.3B8) in ER immunoprecipitates (Pab.H184), similar to panel B. (D) Survey of breast cells. Immunoblots of ER and MGMT in reported breast cell lines are shown: note the absence of the dual ER-positive and MGMT-deficient phenotypes. (E) Immunoblot analysis of SRC-1 and ER in SRC-1 immunoprecipitates from SV-ER (ER MGMT) nuclear extracts, similar to panel B: note that the levels of ER protein were not affected by MeI and 6BG treatments compared to the MCF7 (ER MGMT) cells in panel B. (F) Immunoblot analysis of CBP/p300 (Pab.451) and MGMT (Pab.MGMT) and in CBP/p300 immunoprecipitates (Mab.CBP/p300), similar to panel B.

Article Snippet: Antibodies for CBP/p300 (rabbit polyclonal Pab.451), ER- (Pab.HC20, Pab.H184, and Mab.F10) and SRC-1 (goat Pab.N19 and Pab.M20) were from Santa Cruz.

Techniques: Labeling, Recombinant, Western Blot, Control