The cell lysates were prepared as previously described,[28 ] and the protein concentration was determined with a BCA kit (Thermo Fisher Scientific). Protein samples were separated by sodium dodecyl sulfate (SDS)‐PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 5% fat‐free milk for 1 h, the PVDF membrane was incubated with primary antibody for 1–2 h at room temperature and then washed with 1× tris‐buffered saline with Tween (TBST) for 30 min. Next, the membrane was incubated with the corresponding horseradish peroxidase‐conjugated secondary antibodies for 1 h. After washing with TBST, signals were detected using ECL substrate (Bio–Rad, Hercules, CA, USA). The antibodies used included actin and RanBP3 from Santa Cruz Biotechnology (Santa Cruz, CA, USA), CDK4 and p‐Rb from Cell Signaling Technology (Beverly, MA, USA), cyclin D1, CDK6, GAPDH, histone H3, lamin B1, β‐catenin, and c‐Myc from Proteintech (Rosemont, IL, USA).
Histone h3
Manufactured by Proteintech
Sourced in United States, China, United Kingdom
Histone H3 is a core histone protein that is a fundamental component of chromatin in eukaryotic cells. It plays a crucial role in the structural organization of DNA within the nucleus.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Proteintech (18 mentions)
β actin, by Proteintech (13 mentions)
Pvdf membrane, by Merck Group (10 mentions)
Bcl2 associated x (bax), by Proteintech (6 mentions)
Bcl 2, by Proteintech (4 mentions)
E cadherin, by Proteintech (3 mentions)
γh2ax, by Cell Signaling Technology (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
E cadherin, by Abcam (3 mentions)
Bcl 2, by Abcam (3 mentions)
β actin, by Merck Group (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Bca protein assay, by Thermo Fisher Scientific (3 mentions)
β catenin, by Proteintech (2 mentions)
Cyclin d1, by Proteintech (2 mentions)
α smooth muscle actin α sma, by Proteintech (2 mentions)
Sybr premix ex taq 2, by Takara Bio (2 mentions)
Hif 1α, by Abcam (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Vimentin, by Abcam (2 mentions)
59 protocols using histone h3
1
Western Blot Analysis of Cellular Proteins
2
Antibody Validation and Cellular Signaling Assays
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Antibody against CIP4 (Cat:612556) was purchased from BD Biosciences (NY, USA). Antibodies against GAPDH (Cat:60004), Cdc42 (Cat:10155), Cortactin (Cat:11381), Histone H3 (Cat:17168), FLAG tag (Cat:66008) were purchased from Proteintech (Illinois, USA). Antibodies against p65 (Cat:8242), p-p65 (Cat:3033), the Active Cdc42 Detection Kit (Cat: 8819) were purchased from Cell Signaling Technology (Massachusetts, USA). Antibodies against His tag (Cat:0287R) and GST tag (Cat:33007M) were purchased from Bioss (Beijing, China). The Cdc42 inhibitor ML141 and the NF-κB signaling inhibitor QNZ were purchased from Selleck Chemicals (Shanghai, China). The NF-κB signaling activator LPS was purchased from Sigma (Missouri, USA).
3
Antibodies and Reagents for Cell Culture
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2.1 Antibodies and other reagents DMEM, DMEM-F12 medium and fetal bovine serum (FBS) was from Gibco (Gaithersburg, Maryland). Penicillin and streptomycin were purchased from Invitgen (Carlsbad, CA). N-acetylcysteine (NAC), mannitol came from Sigma (St. Louis, MO). Tempol and TAK242 came from Selleck Chemicals (Houston, Texas). NQO1 pcDNA3.1(+) plasmid and control pcDNA3.1(+) plasmid were bought from GenePharma (Suzhou, China). FuGENEHD transfection reagents were obtained from Promega (Madison, Wisconsin, USA). TRIzol was bought from Invitrogen (Carlsbad, CA). Takara (Shiga, Japan) was the source of SYBR Premix Ex Taq II. The antibodies against α-smooth muscle actin (α-SMA), E-cadherin, collagen IV, bronectin, Nox1, Nox4, TLR4, NQO1, Histone H3, IL-6, TNF-α and β-actin were bought from Proteintech (Chicago, IL), while the antibodies of TGF-β1, NF-κB p65 and MCP-1 came from Abcam (Cambridge, UK). Antibodies against Smad-2, Smad-3, P-Smad2, P-Smad3 were purchased from Cell Signaling Technology (Beverly, MS). Adeno-associated virus serotype 9 (AAV9) was obtained from HanBio Technology (HH20191024HBYXL-AAV01, Shanghai, China).
4
Antibodies for Molecular Signaling Analysis
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β-elemene was obtained from the National Institutes for Food and Drug Control (NIFDC; Beijing, China). Polyclonal antibodies Prx-1, monoclonal antibodies HIF-1α were obtained from Abcam (Cambridge, MA, USA). Monoclonal antibody p65 was obtained from Cell Signaling Technology (Danvers, MA, USA). Monoclonal antibody F/80 was obtained from Santa Cruz (Santa Cruz, CA, USA). Polyclonal antibody MCP-1 was obtained from R&D systems (Minneapolis, MN, USA). Polyclonal antibody Histone-H3, monoclonal antibodies β-actin, MCP-1 were obtained from ProteinTech (Chicago, IL, USA). Fluorescein (FITC)–conjugated Goat Anti-Mouse IgG(H+L) antibody was obtained from ProteinTech (Chicago, IL, USA).
5
Testicular Protein Expression Analysis
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Total testicular protein was extracted and quantified，The proteins (40μg) were separated by sodium dodecyl sulfate (SDS)-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane, which was then blocked in 5% non-fat milk for 1 h. TBST diluted primary antibodies (1:1000) against Nrf2, SOD1, Histone H3, and HO-1 (Proteintech, China); c-Kit, Stra8, 4-hydroxynonenal (4-HNE), NQO1 (1:1000 - 1:3000, Abcam, China); Cleaved-Caspase3, β-actin and γH2AX (1:1000, Cell Signaling Technology, USA); 3-nitrotyrosine (3-NT), PLZF, Phospho-Nrf2-S40 (1:1000, Abclonal, China), were incubated with the membrane at 4◦C overnight and the membrane was washed three times with TBST. Subsequently treated with appropriated second antibody (1:1000, Beyotime, China) for 1 h at room temperature, and the membrane was washed three times with TBST. Then visualized using an enhanced chemiluminescence detection kit. The results were analyzed by FluorChem M (Alpha, United States).
6
Western Blot Analysis of Apoptosis Markers
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Protein lysates from cells were separated by 10% SDS-PAGE, transferred to 0.22-μm NC membranes (Sigma, Shanghai, China) and incubated with anti-hnRNPUL2 rabbit polyclonal antibody (1: 2000; Abcam, Cambridge, UK) or rabbit polyclonal antibodies against caspase-9, cleaved caspase-9, caspase-3 and cleaved caspase-3 (1:500; CST, Danvers, MA, USA). The band intensity was measured by densitometry using the Quantity One Software (Bio-Rad, West Berkeley, CA, USA). The protein levels were normalized with that of tubulin, GAPDH or histone H3 (1: 1000; Proteintech Group Inc., Wuhan, China). All experiments were repeated in triplicate, and the representative results were shown.
7
Western Blot Protein Analysis
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Protein samples isolated as above were used for standard Western blotting experiments. Briefly, 12% SDS-PAGE was used to separate equal protein samples (30 μg each), which were then transferred to PVDF membranes (Millipore, MA, USA). Blots were next blocked for 1 h using 5% non-fat milk in TBST, prior to incubation overnight with antibodies specific for Dishevelled (DVL) (Invitrogen), β-catenin (ThermoFisher), glycogen synthase kinase-3b (GSK-3β) (BIOSS, Beijing, China), (phospho-S9)-GSK-3b (BIOSS), VEGF-A (ThermoFisher), bFGF (AVIVA SYSTEMS BIOLOGY, USA), VE-Cadherin (AVIVA SYSTEMS BIOLOGY), histone H3 (Proteintech, China), and β-actin (Beyotime) (all 1:1000 in TBST) at 4 °C. Blots were then washed thrice using TBST (5 min/wash) prior to an additional 1 h room temperature incubation with biotin-conjugated secondary antibodies (1:1000 in TBST) (Beyotime). A BeyoECL Plus kit (Beyotime Bio) was then used to detect all protein samples based on provided directions, with ImageJ (https://imagej.nih.gov/ij/ ) being used for densitometric analyses. All experiments were repeated a minimum of three times.
8
Protein Expression Analysis by Western Blot
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Cells were washed with cold PBS and homogenized in lysis buffer containing protease inhibitors (keyGEN, Nanjing, China) on ice. After centrifugation, the supernatant containing protein was collected. Total protein and 5 × SDS loading buffer were mixed and boiled at 100 °C for 5 min. Samples were separated by electrophoresis on 10% SDS-polyacrylamide gel and transferred onto polyvinylidene fluoride membranes, after which the membranes were blocked for 1 h at room temperature with 5% skim milk supplemented with 0.1% Tween 20 (TBST). Each membrane was then first incubated overnight with a primary antibody at 4 °C and then with a secondary antibody for 60 min at room temperature. Immunoreactive bands were visualized using a chemiluminescence (ECL) detection system or LI-COR Odyssey infrared imaging system. Primary antibodies are listed below: PITPNC1 (Sigma, Saint Louis, MO, USA), CPT1B, p-AKT (Ser473), MMP9, Cleaved Caspase 3 (Cell Signaling Technology, Danvers, MA, USA), SREBP1 (Novus Biological), CD36, BCL2, BAX, E-cadherin, Vimentin, PPARγ, PPARα/β, Ki67 (Abcam, Cambridge, MA, USA), Histone H3, β-actin (Proteintech, Chicago, USA).
9
Protein Extraction and Western Blot Analysis
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The total proteins of cells were extracted using RIPA buffer (Solarbio) containing protease inhibitor (Solarbio) and phosphatase inhibitor (Bosterbio, Wuhan, China). The cytoplasmic and nuclear proteins of cells were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (Bosterbio). The proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Then, the membranes were blocked with nonfat milk and incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies conjugated with horseradish peroxidase. Finally, the signals on the membranes were detected by enhanced chemiluminescence reagent under an Amersham Imager 600 (General Electric Company, USA), and the grey values of the protein bands were analysed by ImageJ software.
Primary antibodies included the following: PSAT1 (ab96136, Abcam, Cambridge, MA, USA); ALP (ab108337, Abcam); COL1A1 (#84336, CST, Danvers, MA, USA); RUNX2 (ab23981, Abcam); Akt (pan) (#4691, CST); p-Akt (Ser473) (p-Akt) (#4060, CST); p-GSK3β (#9323, CST); GSK-3β (#12456, CST); β-Catenin (#8480, CST); Nonphospho (Active) β-Catenin (#8814, CST); ATF4 (#11815, CST); β-Actin (sc-517582, CST); Histone-H3 (17168-1-AP, Proteintech, Chicago, IL, USA); and GAPDH (HRP-60,004, Proteintech).
Primary antibodies included the following: PSAT1 (ab96136, Abcam, Cambridge, MA, USA); ALP (ab108337, Abcam); COL1A1 (#84336, CST, Danvers, MA, USA); RUNX2 (ab23981, Abcam); Akt (pan) (#4691, CST); p-Akt (Ser473) (p-Akt) (#4060, CST); p-GSK3β (#9323, CST); GSK-3β (#12456, CST); β-Catenin (#8480, CST); Nonphospho (Active) β-Catenin (#8814, CST); ATF4 (#11815, CST); β-Actin (sc-517582, CST); Histone-H3 (17168-1-AP, Proteintech, Chicago, IL, USA); and GAPDH (HRP-60,004, Proteintech).
10
Muscle Protein Extraction and Western Blot Analysis
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Proteins were extracted from muscle tissue and cells using radioimmunoprecipitation assay (RIPA) buffer with 1% (v/v) phenylmethylsulfonyl fluoride (Beyotime Biotechnology, China). Western blotting was performed according to a previously reported method.34 The antibodies used included MyoG (sc‐12732; 1:200; Santa Cruz Biotechnology, USA), MyoD (sc‐760; 1:200; Santa Cruz Biotechnology, USA), MyHC (sc‐376157; 1:1000; Santa Cruz Biotechnology, USA), β‐actin (sc‐4777; 1:1000; Santa Cruz Biotechnology, USA), MEF2C (sc‐365862; 1:200; Santa Cruz Biotechnology, USA), N‐Ras (sc‐31; 1:200; Santa Cruz Biotechnology, USA), Ki67 (ab16667; 1:1000; Abcam, UK), HuR (sc‐5261; 1:200; Santa Cruz Biotechnology, USA), Histone H3 (17168‐1‐AP; 1:1000; Proteintech, USA), α‐tubulin (1E4C11; 1:1000; Proteintech, USA), GFP (50430‐2‐AP; 1:1000; Proteintech, USA), and GAPDH (sc‐47724; 1:1000; Santa Cruz Biotechnology, USA). All protein levels were normalized to that of the housekeeping protein β‐actin, and densitometric quantification of the western blotting bands was performed using ImageJ software.
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