Cells were harvested and incubated in RIPA buffer supplemented with protease and phosphatase inhibitors. Following centrifugation, total protein concentrations in the lysates were determined using Biorad DC Assay kit (Biorad, Hercules, CA). The same amount of total protein lysates were run on an SDS-PAGE gel and transferred to PVDF membrane. After blocking with 5% milk in TBST buffer, membranes were probed with antibodies against Notch1 (Santa Cruz), phospho-S10-Histone H3 (Millipore), PARP (Santa Cruz), and β-actin (Sigma-Aldrich). Detection was done using HRP-conjugated secondary antibodies (Cell signaling, Danvers, MA) and ECL chemiluminesence (GE Healthcare, Piscataway, NJ).
Phospho histone h3 s10
Manufactured by Merck Group
Phospho-histone H3 S10 is a laboratory reagent that is used to detect and measure the phosphorylation of serine 10 on histone H3 protein. Histone H3 phosphorylation is a key marker of cell cycle progression and mitosis.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (2 mentions)
Dc assay kit, by Bio-Rad (1 mentions)
Notch1, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Ha tag, by Merck Group (1 mentions)
Total histone h3, by Cell Signaling Technology (1 mentions)
Phospho akt s473, by Cell Signaling Technology (1 mentions)
Total c myc, by Cell Signaling Technology (1 mentions)
Gfp tag, by Santa Cruz Biotechnology (1 mentions)
Total bad, by Cell Signaling Technology (1 mentions)
Phospho p70s6k t389, by Cell Signaling Technology (1 mentions)
Total 4e bp1, by Cell Signaling Technology (1 mentions)
Gapdh, by Merck Group (1 mentions)
Total p70s6k, by Cell Signaling Technology (1 mentions)
Total akt, by Cell Signaling Technology (1 mentions)
Active caspase 3, by Cell Signaling Technology (1 mentions)
Cyclin b1, by Cell Signaling Technology (1 mentions)
γh2ax, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
5 protocols using phospho histone h3 s10
1
Western Blot Analysis of Cell Lysates
2
Comprehensive Protein Analysis Protocol
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SDS-PAGE and western blotting was carried out using standard methods. The following antibodies were used: PIM1 (12H8, Santa Cruz), PIM1 (A300–313A, Bethyl Laboratories), Actin (ab6276, Abcam), GAPDH (G8795, Sigma), SUMO2 (51–9100, Zymed), His-tag (27-4710-01, GE Healthcare), HA-tag (12CA5, Sigma), Flag-tag (F1804, Sigma), MYC-tag (9E10, Hybridoma supernatant), GFP-tag (sc-8334, Santa Cruz), GST-tag (sc-459, Santa Cruz), total ERK1/2 (ER16, Transduction lab), and phospho S10 Histone H3 (06–570, Millipore). The following antibodies were purchased from Cell Signaling Technology: phospho S62 c-MYC (13748), total c-MYC (5605), total Histone H3 (4499), phospho S112 Bad (5284), total Bad (9239), phospho T37/46 4E-BP1 (2855), total 4E-BP1 (9644), phospho T389 p70S6K (108D2), total p70S6K (49D7), phospho S473 AKT (D9E), total AKT (40D4), phospho T202/Y204 ERK1/2 (9106) and phospho tyrosine-100 (9411). Sheep polyclonal UBC9 and chicken polyclonal RNF4 were from Ron Hay, University of Dundee. Secondary antibodies were purchased from Biorad and Thermo Fisher Scientific.
3
Western Blot and IHC Protein Analysis
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For immunodetection in protein lysates, proteins were separated on SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad), and probed using specific primary antibodies against MYC (Cell Signaling), cyclin B1 (Cell Signaling), P53 (Cell Signaling), P21 (Santa Cruz Biotechnology), and phosphorylated H2AX (γ-H2AX; Millipore 05-636). Actin (1:10,000; Sigma) was used as a loading control. Ki67 (Abcam ab16667), phospho-histone H3 S10 (Millipore 06-570), and active caspase 3 (Cell Signaling 9661) were used for detection of the corresponding antigens in paraffin sections.
4
Cryosectioning and Immunofluorescence of Embryos
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Fixed embryos were embedded in OCT Tissue-Tek (Thermo Fisher Scientific, Waltham, MA), and sectioned (8 μm) using a Leica CM 3050S cryostat (Leica Microsystems, Bannockburn, IL). Sections were blocked in a solution containing 2% horse serum, 0.2% Triton X-100, and 5% bovine serum albumin in phosphate-buffered saline, followed by incubation with primary antibody overnight at 4°C. The following primary antibodies were used: Cypher24 (link) (1:300), CD31 (1:300; BD Biosciences), sarcomeric alpha(α)-actinin (1:300; Sigma-Aldrich), myomesin (1:300; Santa Cruz Biotechnology), cleaved caspase 3 (1:300; Cell Signaling Technology), and phospho-histone H3 S10 (1:300; Millipore). Subsequently, sections were incubated with fluorescently labeled secondary antibodies. Confocal microscopy was performed using a Zeiss Axioplan 2 upright confocal microscope (Carl Zeiss Inc, Thornwood, NY).
5
Antibody Characterization and Recombinant Kinase Assays
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Anti-mouse CD3 (145-2C11) was from American Type Culture Collection while anti-GFP and anti-GST were from Santa Cruz. Anti-SKAP1 (BD Transduction Laboratories), anti-Myc (Cell Signaling), anti-V5 (Invitrogen), anti-FLAG and anti-β-Actin (Sigma) were purchased as assigned. Mouse monoclonal antibodies used in blotting included PLK1 (1:1,000; Cell Signaling), CDK1 (1:1,000; Millipore), Cyclin B1 (1:1,000; Sigma), Cyclin A (1:1000; Cell Signaling), β-Actin (1:200,000; Sigma), phospho-Histone H3 (S10) (1:1,000; Millipore). HRP-conjugated secondary antibodies (1:5,000) were purchased from the The Jackson Laboratory (Maine). Recombinant kinases PLK1, PLK3, CDK1, CDK2, MAPK, Aurora B, CAMK and ZAP-70 were from ProQuinase. Histone H1 protein were obtained from Biolabs).
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