The total protein was extracted from cells by using RIPA reagent containing protease inhibitor PMSF and phosphatase inhibitors NaF and Na3VO4. The cytoplasm and nuclear protein were separated by use of Beyotime cytoplasmic nuclear extraction reagents (Jiangsu, China). The CBA protein assay kit (Beyotime Institute of Biotechnology) was used to detect the intensity levels. The proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes was immersed in a blocking solution TBS-T (Tris-buffered saline with Tween-20) containing 5% non-fat milk for 2 h and then incubated with the primary antibodies at 4°C overnight. The antibodies used were: PTEN and PLCɛ (1: 300 Santa Cruz, CA), AKT (1: 500 WanleiBio, China), P-AKT (S473) (1: 500 Abcam, MA, USA), GAPDH (1: 2000 ImmunoWay, USA), Histone H3 (1: 500 WanleiBio, China). After incubation with horseradish peroxidase-conjugated secondary antibody for 1 h at 37°C, the bands were exposed and developed using an enhanced chemiluminescence kit (Beyotime). All the experiments were performed 3 times.
Histone h3
Manufactured by Wanlei
Sourced in China
Histone H3 is a core histone protein found in eukaryotic cells. It is a key component of the nucleosome, the basic unit of chromatin. Histone H3 plays a crucial role in the organization and regulation of DNA within the cell nucleus.
Automatically generated - may contain errors
Lab products found in correlation
Cleaved caspase 3, by Wanlei (4 mentions)
Cyclin a, by Santa Cruz Biotechnology (3 mentions)
Nf κb p65, by Wanlei (3 mentions)
P akt, by Wanlei (3 mentions)
Rassf1a, by Abcam (3 mentions)
Stat3, by Wanlei (3 mentions)
P stat3, by Wanlei (3 mentions)
Cyclin e, by Santa Cruz Biotechnology (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
Cyclin d1, by Santa Cruz Biotechnology (3 mentions)
Bcl 2, by Wanlei (3 mentions)
P erk, by Wanlei (3 mentions)
Ps127 yap, by Cell Signaling Technology (2 mentions)
Cyclin b1, by Proteintech (2 mentions)
Bcl2 associated x (bax), by Proteintech (2 mentions)
Pakt s473, by Abcam (1 mentions)
Enhanced chemiluminescence kit, by Beyotime (1 mentions)
Gapdh, by Immunoway (1 mentions)
Bax 50599 2 lg, by Proteintech (1 mentions)
Cyclinb1 55004 1 ap, by Proteintech (1 mentions)
8 protocols using histone h3
1
Protein Extraction and Western Blot Analysis
2
Comprehensive Protein Expression Analysis
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Total protein from cells or tissues was extracted and quantified. Twenty‐five micrograms of proteins was separated by 10% polyacrylamide gel electrophoresis, and then transferred onto a nitrocellulose membrane. Skim milk (5%) was used to block non‐specific antigen binding, and then primary antibodies were added (RASSF1A, ab23950, Abcam; YAP, #14074, Cell Signaling Technology; pS127‐YAP, #13008; Cell Signaling Technology; Cyclin A, sc‐596, Santa Cruz Biotechnology; Cyclin B1, 55004‐1‐AP, Proteintech; Cyclin D1, sc‐246, Santa Cruz Biotechnology; Cyclin E, sc‐25303, Santa Cruz Biotechnology; CDK1, WL02373, Wanleibio; cleaved‐caspase‐3, WL02348, Wanleibio; Bcl‐2, WL01556, Wanleibio; BAX, 50599‐2‐lg, Proteintech; p73, WL01604, Wanleibio; p53, sc‐126, Santa Cruz Biotechnology; p21, WL0362, Wanleibio; AKT, WL0003b, Wanleibio; p‐AKT, WLP001a, Wanleibio; ERK, WL01864, Wanleibio; p‐ERK, WLP1512, Wanleibio; STAT3, WL03207, Wanleibio; p‐STAT3, WLP2412, Wanleibio; NF‐κB P65, WL01980, Wanleibio; LATS1/2, YT2543, ImmunoWay Biotechnology; p‐LATS1/2, YP1222, ImmunoWay Biotechnology; MST1/2, 37462, Signalway Antibody; p‐MST1/2, bs‐3294R, Bioss; β‐actin, sc‐47778, Santa Cruz Biotechnology; Histone H3, Wanleibio) and incubated at 4°C overnight. Next, secondary antibodies were added, followed by incubation at room temperature for 1 hour and subsequent exposure to an X‐ray film.
3
Western Blot Analysis of Cellular Proteins
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Total protein was extracted using RIPA lysis buffer containing protease and phosphatase inhibitors (APEXBIO, Houston, TX, USA) and quantitated by BCA assay. Proteins were separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes. After blocking nonspecific binding with 5% nonfat milk in tris-buffered saline with 0.05% Tween 20 (TBST) for 1.5 hr at room temperature, the membrane was stained with an antibody against P-gp (Cat#: WL02395, 1 : 500, Wanlei, Shenyang, Liaoning, China), GR (1 : 5,000, Affinity Biosciences, Cincinnati, OH, USA), DCP1A (Decapping enzyme 1A, Cat#: 22373-1-AP, 1 : 5,000, Proteintech), PNRC2 (Proline-rich nuclear receptor coactivator 2, Cat#: A16549, 1 : 1,000, ABclonal, Wuhan, Hubei, China), UPF1 (Up-frameshift mutant 1, Cat#: 23379-1-AP, 1 : 1,000, Proteintech), Histone H3 (1 : 400, Cat#: WL0984a, Wanlei), or β-actin (Cat#: AC026, 1 : 5,000, ABclonal) at 4°C overnight. After three TBST washes, the blots were incubated with horseradish peroxidase (HRP)-conjugated goat antirabbit IgG (H + L) (Cat#: AS014, 1 : 8,000, ABclonal) for 2 hr at room temperature. The bands were visualized using the enhanced chemiluminescence reagents (Affinity) and were quantified with Image J and normalized by β-actin.
4
Western Blot Analysis of Cellular Proteins
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Total proteins were collected in a lysis buffer containing both phosphatase and protease inhibitors. Protein concentrations was determined using a bicinchoniniacid assay (Solarbio, Beijing). The subsequent steps of Western blotting were following the standard protocols. Antibody against GAPDH was obtained from ProteinTech. Antibodies against YAP, BCL-2, p-YAP (Ser127), p-YAP (Ser397), Cyclin D1, Caspase7, C-MYC were purchased from Cell Signaling Technology(Danvers, MA). Histone H3 and Verteporfin were purchased from Wanleibio (Shenyang, China) and Sigma Aldrich (St Louis, USA), respectively.
5
Western Blot Analysis of EI24 and NF-κB
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Proteins from tissues and cells were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Haimen, China) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF; Beyotime). A total of 40 μg (in 20 μl) of protein from each sample was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and was then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Next, the membranes were incubated with 5% milk at room temperature for 1 h. Then, they were incubated with the appropriate primary antibody overnight at 4 °C and then with a horseradish peroxidase-conjugated secondary antibody (1:5,000; Wanleibio, Shenyang, China). The specific primary antibodies included anti-EI24 (1:1000, Proteintech, Wuhan, China) and anti-NF-κB (P65) (1:500, Wanleibio) antibodies. β-Actin and histone H3 (1:1000, Wanleibio) served as the internal controls. Immune complexes were finally visualized with an enhanced chemiluminescence system (Beyotime).
6
Nucleoprotein Extraction and Western Blotting
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Nucleoprotein was extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, China) according to the manufacturer’s instructions. Total protein was extracted using radioimmunoprecipitation buffer supplemented with the protease inhibitor PMSF. Western blotting was performed as described previously (27 (link)). Each blot was repeated in three separate experiments. Band intensities were measured using Fusion software and the data are presented as relative protein levels normalized to GAPDH or histone H3. The antibodies used in this study were as follows: FoxM1 and P-RAD50 (Cell Signaling Technology, Danvers, MA, USA); Bax, Bcl2, GAPDH, NBS1, P-NBS1, MRE11, P-MRE11, RAD50, ATM, P-ATM, and γH2AX (Abcam, Cambridge, UK); and caspase-3, cleaved caspase-3, and histone H3 (Wanleibio, China).
7
Protein Expression Profiling Assay
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Total protein was extracted and quanti ed. Twenty-ve micrograms of protein was separated by 10% g polyacrylamide gel electrophoresis (PAGE), and then transferred to a nitrocellulose membrane. Skim milk 5% was used to block non-speci c antigen binding, and then primary antibodies were added (RASSF1A, Abcam, ab23950; YAP, Cell Signaling Technology, #4912; Cyclin A, Santa Cruz, sc-596; Cyclin B1, Proteintech, 55004-1-AP; Cyclin D1, Santa Cruz, sc-246; Cyclin E, Santa Cruz, sc-25303; CDK1, Wanleibio, WL02373; cleaved-caspase-3, Wanleibio, WL02348; Bcl-2, Wanleibio, WL01556; BAX, Proteintech, 50599-2-lg; p53, Santa Cruz, sc-126; p21, Wanleibio, WL0362; AKT, Wanleibio, WL0003b; p-AKT, Wanleibio, WLP001a; ERK, Wanleibio, WL01864; p-ERK, Wanleibio, WLP1512; STAT3, Wanleibio, WL03207; p-STAT3, Wanleibio, WLP2412; NF-κB P65, Wanleibio, WL01980; β-actin, Santa Cruz, sc-47778; Histone H3, Wanleibio) and incubated at 4 ℃ overnight. Next, secondary antibodies were added and incubated at room temperature for 1 h, and nally exposed on X-ray lm.
8
Western Blot Analysis of Protein Expression
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Total protein was extracted and quanti ed. Twenty-ve micrograms of protein was separated by 10% g polyacrylamide gel electrophoresis (PAGE), and then transferred to a nitrocellulose membrane. Skim milk 5% was used to block non-speci c antigen binding, and then primary antibodies were added (RASSF1A, Abcam, ab23950; YAP, Cell Signaling Technology, #4912; pS127-YAP, Cell Signaling Technology, #13008; Cyclin A, Santa Cruz, sc-596; Cyclin B1, Proteintech, 55004-1-AP; Cyclin D1, Santa Cruz, sc-246; Cyclin E, Santa Cruz, sc-25303; CDK1, Wanleibio, WL02373; cleaved-caspase-3, Wanleibio, WL02348; Bcl-2, Wanleibio, WL01556; BAX, Proteintech, 50599-2-lg; p73, Wanleibio, WL01604; p53, Santa Cruz, sc-126; p21, Wanleibio, WL0362; AKT, Wanleibio, WL0003b; p-AKT, Wanleibio, WLP001a; ERK, Wanleibio, WL01864; p-ERK, Wanleibio, WLP1512; STAT3, Wanleibio, WL03207; p-STAT3, Wanleibio, WLP2412; NF-κB P65, Wanleibio, WL01980; β-actin, Santa Cruz, sc-47778; Histone H3, Wanleibio) and incubated at 4 °C overnight. Next, secondary antibodies were added and incubated at room temperature for 1 h, and nally exposed on X-ray lm.
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