Analysis of HCC827 cells exposed to 1 μM gefitinib for the indicated times. The phosphorylation status of histone H3 and expression level of β-actin were measured simultaneously using the PhosphoPlus® Histone H3 (Ser10) In-Cell Duet (ICW Compatible). With increased exposure to gefitinib, a significant decrease (~3-8-fold) in phospho-histone H3 signal (green) was observed. Levels of β-actin (red) decreased after 24 hrs of exposure to gefitinib due to cell death. Data and images were generated on the LI-COR® Biosciences Odyssey® Infrared Imaging System.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 μg/ml BSA, and 50% glycerol. Store at –20°C. Do not aliquot either cocktail.
PhosphoPlus® Histone H3 (Ser10) In-Cell Duet from Cell Signaling Technology (CST) provides an easy method to assess protein activation status using a multi-well plate scanner with near infrared detection capabilities, such as the LI-COR® Biosciences Odyssey® Infrared Imaging System. This kit contains a pre-optimized activation state and total protein antibody cocktail, selected based on superior performance. Phosphorylated and total protein are detected simultaneously in the same well, allowing levels of phosphorylated protein to be normalized to total protein. A near infrared detection cocktail is also included.
Phospho-Histone H3 (Ser10) antibody recognizes endogenous levels of histone H3 only when phosphorylated at Ser10. This antibody does not cross-react with other phosphorylated histones or with acetylated histones. β-Actin antibody recognizes endogenous levels of total β-actin protein.
Monoclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser10 of human histone H3 protein or by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human β-actin protein.
Modulation of chromatin structure plays an important role in the regulation of transcription in eukaryotes. The nucleosome, made up of DNA wound around eight core histone proteins (two each of H2A, H2B, H3, and H4), is the primary building block of chromatin (1). The amino-terminal tails of core histones undergo various post-translational modifications, including acetylation, phosphorylation, methylation, and ubiquitination (2-5). These modifications occur in response to various stimuli and have a direct effect on the accessibility of chromatin to transcription factors and, therefore, on gene expression (6). In most species, histone H2B is primarily acetylated at Lys5, 12, 15, and 20 (4,7). Histone H3 is primarily acetylated at Lys9, 14, 18, 23, 27, and 56. Acetylation of H3 at Lys9 appears to have a dominant role in histone deposition and chromatin assembly in some organisms (2,3). Phosphorylation at Ser10, Ser28, and Thr11 of histone H3 is tightly correlated with chromosome condensation during both mitosis and meiosis (8-10). Phosphorylation of Thr3 of histone H3 is highly conserved among many species and is catalyzed by the kinase haspin. Immunostaining with phospho-specific antibodies in mammalian cells reveals mitotic phosphorylation of H3 Thr3 in prophase and its dephosphorylation during anaphase (11).
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PhosphoPlus is a trademark of Cell Signaling Technology, Inc.
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Odyssey is a registered trademark of LI-COR, Inc.