Journal: Molecular Biology of the Cell

Article Title: Estrogen regulates histone deacetylases to prevent cardiac hypertrophy

doi: 10.1091/mbc.e13-08-0444

Figure Lengend Snippet: FIGURE 6: AngII and E2/ERβ regulate HDAC2. (A) HDAC2 mRNA expression is determined by qPCR from cardiomyocytes incubated under various conditions for 24 h. GAPDH is determined as a control. Data are from three experiments. *p < 0.05 for control vs. AngII, +p < 0.05 for AngII vs. AngII + E2 or DPN. (B) HDAC2 protein abundance. *p < 0.05 for control vs. AngII or AngII plus condition in ERβKO mice, +p < 0.05 for AngII vs. AngII + E2 or DPN. (C) HDAC2 S394 phosphorylation in nuclear and cytosolic cell fractions. Total HDAC2 is also shown, and the bar graph is from three experiments. *p < 0.05 for control vs. AngII, +p < 0.05 for AngII vs. AngII + E2 or DPN. (D) CK2 activity in cardiomyocytes. *p < 0.05 for control vs. AngII or ET-1, +p < 0.05 for AngII or ET-1 vs. same + E2, DPN, or TBB. (E) HDAC2 S394 phosphorylation is stimulated by hypertrophic peptides in a CK2-dependent manner and inhibited by E2/ERβ. HDAC2 total protein is shown as loading control. *p < 0.05 for control vs. AngII or ET-1, +p < 0.05 for AngII or ET-1 vs. same+ E2, DPN, or TBB. (F) AngII stimulates CK2 abundance in the nucleus of cardiomyocytes, which is inhibited by E2, DPN, and CK2 inhibitors. Subcellular fractionation of CK2 protein is shown. This study was repeated.

Article Snippet: To the lysates/antibody/bead complexes, 40 μl of stock mixture, made up as 20 μl of 3× kinase buffer (25 mM HEPES, pH 7.5, 10 mM Mg acetate, 2 mM dithiothreitol, 40 mM ATP), and 10 μl of H2O was added to 2 μg of CK2 substrate peptide (RRRADDSDDDDD; Signal Chem, Richmond, Canada) in 9 μl of water with 1 μl of [γ-32P]ATP.

Techniques: Expressing, Incubation, Control, Quantitative Proteomics, Phospho-proteomics, Activity Assay, Fractionation

Journal: Molecular Biology of the Cell

Article Title: Estrogen regulates histone deacetylases to prevent cardiac hypertrophy

doi: 10.1091/mbc.e13-08-0444

Figure Lengend Snippet: FIGURE 8: In vivo regulation of CK2 and HDAC2. (A) CK2 activity was determined by immunoprecipitating the kinase from the ventricular proteins for in vitro assay using substrate protein. Bar graph is from four to six mouse samples per condition in WT and ERβKO mice. *p < 0.05 for control vs. AngII or AngII + condition in ERβKO mice, +p < 0.05 for AngII vs. AngII + E2 or β-LGND2 in WT mice. (B) HDAC2 S394 phosphorylation is stimulated by AngII and is inhibited by E2 and β-LGND2, the latter only in WT mice. Total HDAC2 protein is normalization control. *p < 0.05 for control vs. AngII or AngII + condition in ERβKO mice, +p < 0.05 for AngII vs. AngII + E2 or β-LGND2 in WT mice. (C) HDAC2 protein abundance is stimulated by AngII but inhibited by E2 or β-LGND2 in WT mice. Data analysis is done as in B.

Article Snippet: To the lysates/antibody/bead complexes, 40 μl of stock mixture, made up as 20 μl of 3× kinase buffer (25 mM HEPES, pH 7.5, 10 mM Mg acetate, 2 mM dithiothreitol, 40 mM ATP), and 10 μl of H2O was added to 2 μg of CK2 substrate peptide (RRRADDSDDDDD; Signal Chem, Richmond, Canada) in 9 μl of water with 1 μl of [γ-32P]ATP.

Techniques: In Vivo, Activity Assay, In Vitro, Control, Phospho-proteomics, Quantitative Proteomics