rabbit anti-histone-h3ac  (Active Motif)

Journal: Oncology Letters

Article Title: Sodium valproate affects the expression of p16 INK4a and p21 WAFI/Cip1 cyclin‑dependent kinase inhibitors in HeLa cells

doi: 10.3892/ol.2024.14563

Figure Lengend Snippet: H3Ac and HDAC activity in VPA-treated HeLa cells. (A) Confocal microscopy images. Images are representative of three independent experiments, comprising the analysis of 80 nuclei. Scale bars, 10 µm. (B) Graphs representing FI profiles along the red line drawn in the merged image of selected nuclear images to identify the immunofluorescence signals for H3Ac (green) and TO-PRO-3-stained DNA (blue). (C) Fluorescence intensity of H3Ac signals increased following treatment with 2 mM VPA. (D) Western blotting and respective densitometry of five independent experiments indicated increased abundance of the H3Ac protein following VPA treatment. β-actin was used as a loading control. (E) HDAC enzymatic activity was reduced after cell treatment with 2 mM VPA, evaluated from five independent experiments. Data are presented as the mean ± standard error of the mean. *P<0.05. A.U., arbitrary units; Ctrl, control; FI, fluorescence intensity; H3Ac, histone H3 acetylation; HDAC, histone deacetylase; VPA, valproate.

Article Snippet: The proteins were transferred to nitrocellulose membranes (Thermo Fisher Scientific, Inc.), which were blocked in 4% BSA for 2 h, at 25°C and separately incubated with mouse anti-p16 INK4a , (1:150; cat. no. MA5-17054; Thermo Fisher Scientific, Inc.) mouse anti-p21 WAFI/Cip1 (1:100; cat. no. 1026-MSM11-P1; Thermo Fisher Scientific, Inc.) and rabbit anti-H3Ac (1:4,000; cat. no. PA5-114693; Thermo Fisher Scientific, Inc.) primary antibodies overnight in 1X Tris-buffered saline −0.1% Tween 20 (TBST; cat. no. 91414; Sigma-Aldrich; Merck KGaA) blocking solution at 4°C.

Techniques: Activity Assay, Confocal Microscopy, Immunofluorescence, Staining, Fluorescence, Western Blot, Control, Histone Deacetylase Assay

Journal: International Journal of Molecular Sciences

Article Title: Fluoride Alters Gene Expression via Histone H3K27 Acetylation in Ameloblast-like LS8 Cells

doi: 10.3390/ijms25179600

Figure Lengend Snippet: Increase in histone acetylation (H3ac and H3K27ac) via sodium butyrate in LS8 cells. LS8 cells were treated with 1 mM SB with or without 5 mM NaF for 24 h. ( A ) H3ac (17 kDa) levels were detected via WB. SB increased the protein level of H3ac dose-dependently. β-Actin (44 kDa) was used as a loading control. Representative images are shown. Quantification and statistical analyses of relative protein levels are shown in . ( B ) SB treatment for 24 h increased the signal of H3ac in nuclei compared to control (Ctrl). Nucleus (DAPI; blue), H3ac (red), and Actin (green) were detected via immunofluorescence. Representative images are shown. Scale bars; 50 μm. ( C ) H3K27ac (17 kDa) expressions were detected via WB. SB increased the protein levels of H3ac and H3K27ac with or without fluoride. β-Actin (44 kDa) was used as a loading control. Representative images are shown. Quantification and statistical analyses of relative protein levels are shown in .

Article Snippet: Next, cells were incubated with rabbit anti-Histone H3ac (pan-acetyl) (PA5-116785) (1:200, Thermo Fisher Scientific) or rabbit anti-acetyl-Histone H3 (K27) (#8173) (1:200, Cell Signaling Technology) overnight at 4 °C.

Techniques: Control, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Fluoride Alters Gene Expression via Histone H3K27 Acetylation in Ameloblast-like LS8 Cells

doi: 10.3390/ijms25179600

Figure Lengend Snippet: Increase in histone acetylation (H3ac and H3K27ac) via sodium butyrate in LS8 cells. LS8 cells were treated with 1 mM SB with or without 5 mM NaF for 24 h. ( A ) H3ac (17 kDa) levels were detected via WB. SB increased the protein level of H3ac dose-dependently. β-Actin (44 kDa) was used as a loading control. Representative images are shown. Quantification and statistical analyses of relative protein levels are shown in . ( B ) SB treatment for 24 h increased the signal of H3ac in nuclei compared to control (Ctrl). Nucleus (DAPI; blue), H3ac (red), and Actin (green) were detected via immunofluorescence. Representative images are shown. Scale bars; 50 μm. ( C ) H3K27ac (17 kDa) expressions were detected via WB. SB increased the protein levels of H3ac and H3K27ac with or without fluoride. β-Actin (44 kDa) was used as a loading control. Representative images are shown. Quantification and statistical analyses of relative protein levels are shown in .

Article Snippet: Primary Ab: rabbit anti-phospho-HDAC2 (Ser394) (#69238), rabbit anti-phospho-HDAC3 (Ser424) (#3815), rabbit anti-phospho-HDAC7 (Ser155) (#3443), rabbit anti-acetyl-Histone H3 (K27) (#8173), mouse anti-Histone H3 (#14269), mouse anti-α-tubulin (#3873), mouse anti-β-actin (#3700) (1:1000, Cell Signaling Technology, Boston, MA, USA), rabbit anti-Histone H3ac (pan-acetyl) (PA5-116785) (1:1000, Thermo Fisher Scientific), and rabbit anti-phospho-SIRT1 (Ser47) (BS-3393R) (1:1000, Bioss, Woburn, MA, USA).

Techniques: Control, Immunofluorescence

Journal: eLife

Article Title: The nutrient-sensing GCN2 signaling pathway is essential for circadian clock function by regulating histone acetylation under amino acid starvation

doi: 10.7554/eLife.85241

Figure Lengend Snippet: ( A ) Chromatin immunoprecipitation (ChIP) assay showing that deletion of cpc-3 or cpc-1 did not dramatically affect WC-2 binding at the promoter of frq gene without 3-aminotriazole (3-AT; n = 3; WT: p = 2.60E−07, cpc-3 KO : p = 0.0016, cpc-1 KO : p = 1.06E−05, RAIN; WT vs cpc-3 KO : mesor p = 0.4797, amplitude p = 0.0238, phase p = 0.7395; WT vs cpc-1 KO : mesor p = 0.1151, amplitude p = 0.1311, phase p = 0.0039, CircaCompare). Samples were grown for the indicated number of hours in DD prior to harvesting and processing for ChIP using WC-2 antibody. Occupancies were normalized by the ratio of ChIP to Input DNA. Error bars indicate standard deviation ( n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; Student’s t test was used. Western blot assay showing that WCC was hypophosphorylated in the cpc-3 KO ( B ) and cpc-1 KO ( C ) strains in the presence of 3 mM 3-AT. Proteins were extracted by protein extraction buffer with PPase inhibitors and loaded in each protein lane of 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels containing a ratio of 149:1 acrylamide/bisacrylamide. The faster mobility shift indicated hypophosphorylation levels of WC-1 and WC-2. ( D ) ChIP assay showing slightly decreased histone H3ac levels at the promoter of frq gene in the cpc-1 KO strain without 3-AT at the indicated time points in DD ( n = 3; WT: p = 0.0025, cpc-1 KO : p > 0.05). Error bars indicate standard deviations ( n = 3). *p < 0.05; Student’s t test was used. Figure 2—figure supplement 1—source data 1. Chromatin immunoprecipitation (ChIP) analysis dataset in . Figure 2—figure supplement 1—source data 2. Scan of western blot probed for phosphorylation of WC-1 and WC-2 proteins in WT and cpc-3 KO strains with 3-aminotriazole (3-AT) in . Figure 2—figure supplement 1—source data 3. Scan of western blot probed for phosphorylation of WC-1 and WC-2 proteins in WT and cpc-1 KO strains with 3-aminotriazole (3-AT) in . Figure 2—figure supplement 1—source data 4. Chromatin immunoprecipitation (ChIP) analysis dataset in .

Article Snippet: Antibody , Rabbit polyclonal anti-Histone H3ac , Millipore , Cat# 06-599 , 1:3000.

Techniques: Chromatin Immunoprecipitation, Binding Assay, Standard Deviation, Western Blot, Protein Extraction, Polyacrylamide Gel Electrophoresis, SDS Page, Mobility Shift, Phospho-proteomics

Journal: eLife

Article Title: The nutrient-sensing GCN2 signaling pathway is essential for circadian clock function by regulating histone acetylation under amino acid starvation

doi: 10.7554/eLife.85241

Figure Lengend Snippet: Chromatin immunoprecipitation (ChIP) assay showing that amino acid starvation slightly increased histone H2B levels ( A ) and dramatically decreased histone H3ac levels ( B ) at the promoter of frq gene in the WT strain at DD18 at the indicated concentration of 3-aminotriazole (3-AT). Relative H3ac levels were normalized with H2B levels. ( C, D ) ChIP assay showing that amino acid starvation slightly increased histone H2B levels ( n = 3; WT: p = 3.85E−04, cpc-1 KO : p = 0.0364, RAIN; WT vs cpc-1 KO : mesor p = 0.0312, amplitude p = 0.2155, phase p = 0.2995, CircaCompare) ( C ) and dramatically decreased histone H3ac levels ( n = 3; WT: p = 0.0168, cpc-1 KO : p > 0.05) ( D ) at the promoter of frq gene in the cpc-1 KO strain at the indicated time points in DD. Error bars indicate standard deviations ( n = 3). *p < 0.05; ***p < 0.001; Student’s t test was used. Figure 3—source data 1. Chromatin immunoprecipitation (ChIP) analysis dataset in . Figure 3—source data 2. Chromatin immunoprecipitation (ChIP) analysis dataset in . Figure 3—source data 3. Chromatin immunoprecipitation (ChIP) analysis dataset in . Figure 3—source data 4. Chromatin immunoprecipitation (ChIP) analysis dataset in .

Article Snippet: Antibody , Rabbit polyclonal anti-Histone H3ac , Millipore , Cat# 06-599 , 1:3000.

Techniques: Chromatin Immunoprecipitation, Concentration Assay

Journal: eLife

Article Title: The nutrient-sensing GCN2 signaling pathway is essential for circadian clock function by regulating histone acetylation under amino acid starvation

doi: 10.7554/eLife.85241

Figure Lengend Snippet: ( A ) Race tube assay showing that the conidiation rhythm in gcn-5 KO strain was lost compared with WT strain. ( B ) Luciferase assay showing that the luciferase activity rhythm was impaired in the gcn-5 KO strain after 1 day transition from light to dark. A FRQ-LUC translational fusion construct was expressed in WT and gcn-5 KO strains, and the luciferase signal was recorded in DD for more than 7 days. Normalized data with the baseline luciferase signals subtracted are shown. ( C ) Western blot assay showing that rhythmic expression of FRQ protein was dampened in the gcn-5 KO strain ( n = 3; WT: p = 5.00E−08, gcn-5 KO : p = 0.0016, RAIN; WT vs gcn-5 KO : mesor p = 0.1421, amplitude p = 0.0774, phase p = 0.4319, CircaCompare). RT-qPCR analysis showing that rhythmic expression of frq mRNA was dampened in the gcn-5 KO strain without 3-aminotriazole (3-AT; n = 3; WT: p = 8.37E−06, gcn-5 KO : p > 0.05) ( D ) or with 3-AT ( n = 3; WT: p = 5.39E−12, gcn-5 KO : p > 0.05) ( E ). ( F ) Chromatin immunoprecipitation (ChIP) assay showing decreased histone H3ac levels at the promoter of frq gene in the gcn-5 KO strain at the indicated time points in DD ( n = 3; WT: p = 8.10E−05, gcn-5 KO : p > 0.05). Relative H3ac levels were normalized with H2B levels. ( G ) ChIP assay showing decreased WC-2 levels at the promoter of frq gene in the gcn-5 KO strain at the indicated time points in DD ( n = 3; WT: p = 0.0003, gcn-5 KO : p = 0.0459, RAIN; WT vs gcn-5 KO : mesor p = 0.2939, amplitude p = 0.0010, phase p = 0.6933, CircaCompare). Error bars indicate standard deviations ( n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; Student’s t test was used. Figure 5—source data 1. LumiCycle analysis dataset in . Figure 5—source data 2. Scan of western blot probed for FRQ protein and quantification dataset in . Figure 5—source data 3. RT-qPCR analysis dataset in . Figure 5—source data 4. RT-qPCR analysis dataset in . Figure 5—source data 5. Chromatin immunoprecipitation (ChIP) analysis dataset in . Figure 5—source data 6. Chromatin immunoprecipitation (ChIP) analysis dataset in .

Article Snippet: Antibody , Rabbit polyclonal anti-Histone H3ac , Millipore , Cat# 06-599 , 1:3000.

Techniques: Luciferase, Activity Assay, Construct, Western Blot, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation

Journal: eLife

Article Title: The nutrient-sensing GCN2 signaling pathway is essential for circadian clock function by regulating histone acetylation under amino acid starvation

doi: 10.7554/eLife.85241

Figure Lengend Snippet: ( A ) Race tube assay showing that high concentrations of 3-aminotriazole (3-AT) treatment elongated circadian conidiation period of WT strain. ( B ) Race tube assay showing that the high concentrations of Trichostatin A (TSA) treatment shortened circadian conidiation period of WT strain. ( C ) TSA treatment rescued prolonged circadian period of WT strain caused by 3-AT treatment. WT strain was grown on the race tube medium containing 7.5 mM 3-AT and indicated concentrations of TSA in DD. ( D ) Chromatin immunoprecipitation (ChIP) assay showing that H3ac levels were decreased in gcn-5 KO strains and increased in hda-1 KO strains at the promoter of frq gene. Error bars indicate standard deviations ( n = 3). *p < 0.05; **p < 0.01; Student’s t test was used. ( E ) The hda-1 KO strain exhibited near normal circadian period in the presence of 3-AT. hda-1 KO strains were grown on the race tube medium containing the indicated concentrations of 3-AT in DD. ( F ) TSA treatment rescued the impaired circadian rhythm of cpc-3 KO strain caused by 3-AT treatment. cpc-3 KO strains were grown on the race tube medium containing 3 mM 3-AT and indicated concentrations of TSA in DD. Figure 6—source data 1. RT-qPCR analysis dataset in .

Article Snippet: Antibody , Rabbit polyclonal anti-Histone H3ac , Millipore , Cat# 06-599 , 1:3000.

Techniques: Chromatin Immunoprecipitation, Quantitative RT-PCR

Journal: eLife

Article Title: The nutrient-sensing GCN2 signaling pathway is essential for circadian clock function by regulating histone acetylation under amino acid starvation

doi: 10.7554/eLife.85241

Figure Lengend Snippet: Under normal conditions, CPC-1 is expressed at its basal levels in the WT (Left) or cpc-3 KO (Right) strain, which is required for rhythmic expression of frq gene by recruiting the histone acetyltransferase GCN-5 containing SAGA complex to the frq promoter. Under amino acid starvation conditions, the chromatin in the frq promoter of the WT strain is constitutively compacted (due to decreased H3ac), likely due to activation of histone deacetylases or inhibition of histone acetyltransferases. CPC-3 and CPC-1 signaling pathway was activated by amino acid starvation and the elevated CPC-1 protein would efficiently recruit the histone acetyltransferase GCN-5 containing SAGA complex to promote the histone acetylation levels, which permitted rhythmic WCC binding at the frq promoter (Left). Disruption of the CPC-3 and CPC-1 signaling pathway resulted in decreased histone acetylation levels of the frq gene promoter, reduced WCC binding and damped circadian oscillations in response to the amino acid starvation stress (Right).

Article Snippet: Antibody , Rabbit polyclonal anti-Histone H3ac , Millipore , Cat# 06-599 , 1:3000.

Techniques: Expressing, Activation Assay, Inhibition, Binding Assay, Disruption

Journal: eLife

Article Title: The nutrient-sensing GCN2 signaling pathway is essential for circadian clock function by regulating histone acetylation under amino acid starvation

doi: 10.7554/eLife.85241

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit polyclonal anti-Histone H3ac , Millipore , Cat# 06-599 , 1:3000.

Techniques: Sequencing, Northern Blot, Software

Journal: The EMBO Journal

Article Title: Hypoxia promotes osteogenesis by facilitating acetyl‐CoA ‐mediated mitochondrial–nuclear communication

doi: 10.15252/embj.2022111239

Figure Lengend Snippet:

Article Snippet: Rabbit anti‐histone H3ac, Polyclonal (1:500) , Active Motif , #39139.

Techniques: Transgenic Assay, Isolation, Recombinant, Generated, Software, Bicinchoninic Acid Protein Assay, RNA Extraction, Reverse Transcription, Ligation, Mass Spectrometry, Liquid Chromatography, Microscopy