Antibodies to E-cadherin, acetylated histone H3 (Lys-9), total histone H3, and cleaved caspase-3 were purchased from Cell Signaling Technology (Beverly, MA). Anti-β-catenin was purchased from EMD Millipore (Billerica, MA) and HRP-linked secondary antibodies were purchased from Life Technologies.
Total histone h3
Manufactured by Cell Signaling Technology
Sourced in United States
Total histone H3 is a lab equipment product that measures the total amount of histone H3 protein in a sample. Histone H3 is a core histone protein that plays a fundamental role in the structure of chromatin and the regulation of gene expression.
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Lab products found in correlation
H3k27me3, by Cell Signaling Technology (3 mentions)
Hdac4, by Cell Signaling Technology (2 mentions)
Total bad, by Cell Signaling Technology (2 mentions)
Chemidoc xrs imager, by Bio-Rad (2 mentions)
Supersignal west pico chemiluminescence system, by Thermo Fisher Scientific (2 mentions)
Dot1l, by Cell Signaling Technology (2 mentions)
Dimethyl histone h3 lys79, by Abcam (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti β catenin, by Merck Group (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
C digit blot scanner, by LI COR (1 mentions)
Nitrocellulose membrane, by Santa Cruz Biotechnology (1 mentions)
Acetyl histone h3, by Merck Group (1 mentions)
Gapdh, by Applied Biological Materials (1 mentions)
Lamin b1, by Abcam (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Acetylated histone h3, by Active Motif (1 mentions)
Total histone h4, by Active Motif (1 mentions)
α tubulin, by Merck Group (1 mentions)
25 protocols using total histone h3
1
Immunoblotting for Cell Signaling
2
Protein Extraction and Western Blotting Protocol
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A detailed description of protein extraction and Western blotting procedures can be found in our previous report [5 (link)].
In brief, muscle samples were loaded and separated on a 10% polyacrylamide gel, followed by transfer to a nitrocellulose membrane (Santa Cruz Biotechnology, Inc., Sanford, ME, USA, #sc-3724), after which membranes were incubated in a blocking buffer (TBS-T: 4% non-fat milk powder; Tris-buffered saline, pH 7.4; and 0.1% Tween 20). The membranes were then incubated with primary and secondary antibodies and washed in TBS-T. The primary antibodies used were GAPDH (1:10,000, Applied Biological Materials Inc., Richmond, BC, Canada, # G041), Lamin B1 (1:500, Abcam, Cambridge, MA, USA, # ab16048), MEF2-D (1:1000, EMD Millipore, Temecula, CA, USA, # AB2263), acetyl-Histone H3 (1:1000, EMD Millipore, Temecula, CA, USA, # 06-599), total Histone H3 (1:1000, Cell Signaling Technology, Danvers, MA, USA, # 9715), HDAC4 (1:500, Cell Signaling, Danvers, MA, USA, #2072), HAT P300 (1:500, Abcam, Cambridge, MA, USA, # ab231010).
Secondary HRP-conjugated antibodies (1:30,000) to rabbit or mouse immunoglobulins were from Santa Cruz Biotechnology, CA, USA. Protein bands were detected and quantified using Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA, #170-5061) and C-DiGit Blot Scanner (LI-COR Biotechnology, Lincoln, NE, USA).
In brief, muscle samples were loaded and separated on a 10% polyacrylamide gel, followed by transfer to a nitrocellulose membrane (Santa Cruz Biotechnology, Inc., Sanford, ME, USA, #sc-3724), after which membranes were incubated in a blocking buffer (TBS-T: 4% non-fat milk powder; Tris-buffered saline, pH 7.4; and 0.1% Tween 20). The membranes were then incubated with primary and secondary antibodies and washed in TBS-T. The primary antibodies used were GAPDH (1:10,000, Applied Biological Materials Inc., Richmond, BC, Canada, # G041), Lamin B1 (1:500, Abcam, Cambridge, MA, USA, # ab16048), MEF2-D (1:1000, EMD Millipore, Temecula, CA, USA, # AB2263), acetyl-Histone H3 (1:1000, EMD Millipore, Temecula, CA, USA, # 06-599), total Histone H3 (1:1000, Cell Signaling Technology, Danvers, MA, USA, # 9715), HDAC4 (1:500, Cell Signaling, Danvers, MA, USA, #2072), HAT P300 (1:500, Abcam, Cambridge, MA, USA, # ab231010).
Secondary HRP-conjugated antibodies (1:30,000) to rabbit or mouse immunoglobulins were from Santa Cruz Biotechnology, CA, USA. Protein bands were detected and quantified using Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA, #170-5061) and C-DiGit Blot Scanner (LI-COR Biotechnology, Lincoln, NE, USA).
3
Histone Extraction and Western Blot Analysis
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Histones were extracted following a published protocol through sulphuric acid extraction and TCA-precipitation [[43 (link)]]. One μg of each sample was used for western blot analysis with 15% SDS-PAGE gels and PVDF membranes (Merck Millipore, Berlin, Germany) according to the previously-described protocol. The detection of acetylated and non-acetylated histones was performed with primary antibodies against acetylated histone H3 (1:2,000, #39139, Active Motif, La Hulpe, Belgium), total histone H3 (1:1,000, #3638, Cell Signaling Technology, Inc., Danvers, MA), acetylated histone H4 (1:1,000, #39243, Active Motif, La Hulpe, Belgium) and total histone H4 (1:500, #39269, Active Motif, La Hulpe, Belgium).
4
Immunoblotting and Immunoprecipitation Assay Protocol
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Immunoblotting and immunofluorescence staining were carried out as previously described (Jing et al., 2011 ). Immunoprecipitation was performed using Dynabeads Protein A Immunoprecipitation Kit according to the manufacturer's protocol (#10006D, Thermofisher, USA). Antibodies used in this study: HDAC4 (#7628, Cell signaling, Germany), FoxO1 (#2880, Cell signaling, Germany), Pdx1 (sc‐14664, Santa Cruz, USA), acetylated lysine (#9441, Cell signaling Germany), α‐Tubulin (#T6199, Sigma, USA), and total histone H3 (#4499, Cell signaling, Germany) (Table S4 ).
We used GraphPad Prism 5.0 software (GraphPad Software). Differences between three or more groups were assessed by 1‐way ANOVA with Dunnett's correction. p < 0.05 were considered significant.
We used GraphPad Prism 5.0 software (GraphPad Software). Differences between three or more groups were assessed by 1‐way ANOVA with Dunnett's correction. p < 0.05 were considered significant.
5
Histone Modification Analysis in Aorta
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Protein extraction and western blotting were performed as described previously (12 (link)). H3K4me1, H3K9me3, H3K56ac and total histone H3 antibodies were purchased from Cell Signaling Technology. Pooled aortic histone extracts from three mice were subjected to the assay.
6
Western Blot Analysis of Cell Signaling
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20 to 50 μg of protein was diluted 1:1 with 2× loading buffer (100 mM Tris–HCL pH 6.8, 20% glycerol, 4% SDS, 200 mM DTT and Bromophenol Blue), and proteins were denatured for 5 min at 95°C. Samples were resolved on a SDS–PAGE following standard procedures.
Gels were transferred onto Nitrocellulose membrane (GE Healthcare) for 1.5 h at 400 mA. The membrane was then blocked with 5% Milk in TBS‐tween buffer (20 mM Tris pH 7.6, 150 mM NaCl, 0.1% Tween) for 20 min. Membranes were incubated 30 min/1 h or O/N at 4°C in primary antibodies [Cleaved Notch1 (1:1,000, Cell Signalling #4147); β‐Actin (1:10,000, Proteintech #66009‐1‐Ig); Fbxw7 (1:1,000; Abcam #171961); GFP (1:1,000, Cell Signalling #2956); Cdk1 (1:1,000, Cell Signalling #9116); Cdk2 (1:1,000, Cell Signalling #2546); Cdk4 (1:1,000, Santa Cruz #260); Cdk6 (1:1,000, Santa Cruz #177); Cdk8 (1:1,000, Bethyl #A302‐501A‐T); Cyclin A2 (1:1,000, Cell Signalling #4656); Cyclin B1 (1:1,000, Cell Signalling #4138); Cyclin C (1:1,000, Bethyl #A301‐989A‐M); Cyclin D1 (1:5,000, Abcam #137875); Cyclin E1 (1:1,000, Cell Signalling #4129); Total histone H3 (1:1,000, Cell Signalling #9715); Phospho‐histone H3 (1:1,000, Cell Signalling #3377)].
The membranes were then washed with TBS‐Tween and incubated with the appropriate secondary HRP antibody (Cell Signalling, #7074, #7076). After washing, membranes were developed using ECL solution (Pierce).
Gels were transferred onto Nitrocellulose membrane (GE Healthcare) for 1.5 h at 400 mA. The membrane was then blocked with 5% Milk in TBS‐tween buffer (20 mM Tris pH 7.6, 150 mM NaCl, 0.1% Tween) for 20 min. Membranes were incubated 30 min/1 h or O/N at 4°C in primary antibodies [Cleaved Notch1 (1:1,000, Cell Signalling #4147); β‐Actin (1:10,000, Proteintech #66009‐1‐Ig); Fbxw7 (1:1,000; Abcam #171961); GFP (1:1,000, Cell Signalling #2956); Cdk1 (1:1,000, Cell Signalling #9116); Cdk2 (1:1,000, Cell Signalling #2546); Cdk4 (1:1,000, Santa Cruz #260); Cdk6 (1:1,000, Santa Cruz #177); Cdk8 (1:1,000, Bethyl #A302‐501A‐T); Cyclin A2 (1:1,000, Cell Signalling #4656); Cyclin B1 (1:1,000, Cell Signalling #4138); Cyclin C (1:1,000, Bethyl #A301‐989A‐M); Cyclin D1 (1:5,000, Abcam #137875); Cyclin E1 (1:1,000, Cell Signalling #4129); Total histone H3 (1:1,000, Cell Signalling #9715); Phospho‐histone H3 (1:1,000, Cell Signalling #3377)].
The membranes were then washed with TBS‐Tween and incubated with the appropriate secondary HRP antibody (Cell Signalling, #7074, #7076). After washing, membranes were developed using ECL solution (Pierce).
7
Phosphorylation Pathways in Cell Signaling
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Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were from Life Technologies, Inc. (Grand Island, NY). Antibodies against β-actin, phosphorylated RSK2 (Thr577) and His G were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The antibody against Xpress was from Invitrogen Corporation (Carlsbad, CA). The 10X kinase buffer and antibodies against RSK2, phosphorylated histone H3 (Ser10), and total histone H3 were from Cell Signaling Technology, Inc. (Beverly, MA). Active RSK2 was from Upstate Biotechnology, Inc. (Charlottesville, VA) and EGCG was a generous gift from Dr. Chi Tang Ho (Rutgers University, Piscataway, NJ).
8
Epigenetic Regulation and Cell Signaling
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Antibodies: EZH2 (Cell Signaling Technology 3147), Actin (MAB1501), Total Histone H3 (Cell Signaling Technology 3638), H3K27me3 (Cell Signaling Technology 9733), H3K27me2 (Cell Signaling Technology 9728), MLC2 (Cell Signaling Technology 3672), pMLC2 (ser 19) (Cell Signaling Technology 3671), E-Cadherin (BD Biosciences 610182), and Vimentin (Epitomics 4211–1).
9
Prostate Cancer Cell Culture Protocol
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The Myc-CaP (Myc-CaP/AS) cell line [21 (link)] was a kind gift from Dr. Charles Sawyers. Both Myc-CaP/AS and Myc-CaP/CR [33 (link)] cell lines and were cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C, 5% CO2. TRAMP C2 cell lines were a kind gift from Dr. Barbara Foster. TRAMP C2 cell lines were cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C, 5% CO2. LnCaP cell lines were purchased from ATCC, and cultured in RPMI medium (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C, 5% CO2. Primary antibodies towards Ezh2, GAPDH, H3K27me3, Total Histone H3, LC3 and p-γH2AX, activated caspase-3 were purchased from Cell Signaling. Ki-67 was purchased from Thermo Scientific. Etoposide (Sigma-Aldrich) and GSK126 (Xcess Biosciences Inc.) were maintained in DMSO at 1mM and 10mM stock concentrations respectively. Synthetic androgen (R1881) (Toronto Research Chemicals) was maintained at a 10mM stock in 100% ethanol. DZNep (Cayman Chemicals) was maintained in DMSO (10mg/ml) and diluted in PBS (1mg/ml) before use. Etoposide was obtained from the Roswell Park Cancer Institute Pharmacy Department (20mg/ml), and was diluted in PBS (2mg/ml) before use.
10
Western Blot Analysis of Epigenetic Regulators
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Cells were lysed in radioimmunoprecipitation assay buffer (Merck Millipore) supplemented with cOmplete protease inhibitor (Roche) and PhosSTOP phosphatase inhibitor (Roche). 20 µg of protein lysate was mixed with Laemmli sample buffer (Bio-Rad) and β-mercaptoethanol (Sigma-Aldrich) before being run on a 4–12% Novex Bis-Tris polyacrylamide gel (Thermo Fisher Scientific). Blots were transferred to polyvinylidene fluoride membrane (Thermo Fisher Scientific) and blocked with 5% milk (Thermo Fisher Scientific) or 5% BSA (New England Biolabs) in phosphate-buffered saline with 0.1% Tween (Boston Bioproducts) and probed with the following antibodies: Total histone H3 (1:1,000, no. 4499; Cell Signaling Technology), Tri-methyl-histone H3 Lys 27 (1:1,000; no. 07-449; Merck Millipore), EZH2 (1:1,000; no. 5246; Cell Signaling Technology), SUZ12 (1:1,000; no. 3737; Cell Signaling Technology), TRAP1 (1:1,000; no. 612344; BD Biosciences), β-tubulin (1:1,000; no. 2128; Cell Signaling Technology). Secondary detection of horseradish peroxidase–linked antibodies (Cell Signaling Technology) with horseradish peroxidase substrate (Thermo Fisher Scientific) was visualized using Amersham Imager 600 (GE Healthcare Life Sciences). All antibodies were validated using the shRNA knockdown or overexpression experiments shown in this manuscript.
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