Phospho histone h3 serine 10

Manufactured by Cell Signaling Technology

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Phospho-Histone-H3-Serine 10 is a lab reagent that detects phosphorylation of histone H3 at serine 10. It is used to identify cells undergoing mitosis.

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Close spelling variants of this product

Histone H3 (504 citations) Phospho-histone H3 (81 citations) Phospho-histone H3 (Serine 10) antibody (2 citations)

3 protocols using phospho histone h3 serine 10

Differential Protein Expression Analysis

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The cytoplasm and nuclear protein extracts were run on 4–15% Tris-glycine gels (Bio-Rad) and transferred to nitrocellulose membranes (GE). The proteins of interest were detected by incubating with primary antibodies such as NFkB-P65(C-20)(Cat.no:SC-372), IKKα (Cat.no:SC-7218), IKKβ (Cat.no:SC-7329), RSK1(Cat.no:SC-130870), MSK1(Cat.no:SC-25417), H3-Histone(Cat.no:SC-8654), SPT5(Cat.no:SC-28678), GAPDH(Cat.no:SC-25778), Lamin A/C(Cat.no:SC-7292), Phospho-RSK1(Thr348)(Cat.no:SC-101770), Phospho-p65(Ser 276) (Cat.no: SC-101749) were purchased from Santa Cruz Biotechnology. Antibodies against p65 (phospho-S536; Cat no-3033), phospho-MSK1 (Thr 581) (Cat no-9595), Phospho-IKKα (Ser 176/180)(Cat no-2697) and phospho-Histone-H3-Serine 10(Cat no-12201) were purchased from Cell Signaling. Antibodies against Phospho-IKKβ (Tyr 199) (Cat no-PA5–35838) were purchased from life technologies. The appropriate secondary antibodies were applied after 3 washes with 0.05% Tween 20 in phosphate or Tris buffered saline (PBST/TBST). The blots were incubated with substrate ECL (Santa Cruz) prior to exposure to X-ray film (Fuji). GAPDH, Lamin A/C, Histone H3 and SPT5 were used as loading controls for cytoplasmic and nuclear protein fractions. Densitometric analysis on the western blots was performed using NIH-Image J software.

Sahu G., Farley K., El-Hage N., Aiamkitsumrit B., Fassnacht R., Kashanchi F., Ochem A., Simon G.L., Karn J., Hauser K.F, & Tyagi M. (2015). Cocaine promotes both initiation and elongation phase of HIV-1 transcription by activating NF-κB and MSK1 and inducing selective epigenetic modifications at HIV-1 LTR. Virology, 483, 185-202.

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CDK5 Kinase Assay and Mutagenesis

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All reagents and chemicals were of analytical grade. The purified recombinant CDK5:p35 complex (Catalog#14-477) was purchased from Millipore-Sigma. Recombinant shrimp alkaline phosphatase (rSAP) was from New England Biolabs (NEB). Protease and phosphatase inihibitors were from Fisher Scientific and Sigma, respectively. ProLong Gold Antifade Mountant with DAPI was from ThermoFisher Scientific. Antibodies against CDK5 (Cat#sc-6247) and p35 (Cat#sc-820) were from Santa Cruz Biotechnology. Antibodies against Tubulin (Cat#2128), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cat#5174) and phospho-Histone H3 Serine 10 (Cat#53348) were from Cell Signaling. IRDye 680RD and IRDye 800CW secondary antibodies for western blotting were from Li-COR Biosciences and goat anti-rabbit Alexafluor-647 conjugated antibody for immunofluorescence was from Invitrogen. CDK5 (WT and D144N) and p35-HA constructs were gifts from David S. Park (University of Ottawa, Ottawa) and Edward Giniger (NIH/NINDS), respectively. Point mutants were generated using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies). The mutations and the integrity of the rest of the insert were confirmed by sequencing.

Roach B.L., Ngo J.M., Limso C., Oloja K.B, & Bhandari D. (2018). Identification and characterization of a novel phosphoregulatory site on cyclin-dependent kinase 5. Biochemical and biophysical research communications, 504(4), 753-758.

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Protein Extraction and Western Blot Analysis

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Cells were washed in PBS and lysed in the following buffer: 50 mM Tris–HCl, pH 7.4, 0.5% IGEPAL (Sigma‐Aldrich, St. Louis, MO, USA), 150 mM NaCl, 1 mM DTT, 5% glycerol, 50 mM NaF, 1 mM PMSF, 25 mM β‐glycerophosphate, 1 mM vanadate, Complete Mini Protease Inhibitor Cocktail (Roche Diagnostics, Basel, Switzerland). Proteins were then resolved by SDS–PAGE and transferred onto a nitrocellulose membrane. Primary antibodies were directed against α‐tubulin (clone DM1A, 1:5,000; Sigma‐Aldrich, St. Louis, MO, USA), γ‐tubulin (clone GTU‐88, 1:5,000; Sigma‐Aldrich, St. Louis, MO, USA), GFP (ab290; 1:5,000; Abcam, Cambridge, UK), STIL (ab89314, 1:2,000; Abcam, Cambridge, UK), Sas‐6 (Kleylein‐Sohn et al, 2007), CP110 (1:2,000, Schmidt et al, 2009), cyclin A (1:2,000, Maridor et al, 1993), cyclin B1 (05‐373, 1:2,000, Merck Millipore, Darmstadt, Germany), and phospho‐histone H3/serine 10 (3377, 1:1,000, Cell Signaling Technology, Danvers, MA, USA); secondary antibodies were HRP‐conjugated anti‐mouse immunoglobulin (170‐6516, 1:3,000, Bio‐Rad, Hercules, CA, USA) or anti‐rabbit immunoglobulin (170‐6515, 1:3,000, Bio‐Rad, Hercules, CA, USA).

Bauer M., Cubizolles F., Schmidt A, & Nigg E.A. (2016). Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging. The EMBO Journal, 35(19), 2152-2166.

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