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. 2010 Jul 8;5(7):e11489. doi: 10.1371/journal.pone.0011489

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Re et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Purity assessment of nuclei preparations used in functional assays by bright field microscopy and Western blotting. Left panel: Photomicrograph of a typical nuclei preparation following trypan blue staining. Scale bar = 10 µm. Insert: digital magnification of a single nucleus showing the presence of nucleoles (arrow). Right panel: Immunodetection of the membrane and nuclear markers clathrin and lamin A/C respectively in membrane and isolated nuclei fractions from FLAG-GnRH-R-transfected HEK 293 cells. (B) Time course of Histone H3-Lys9/Lys14 acetylation and -Ser10 phosphorylation in GnRH-stimulated nuclei by Western blot analysis. A representative Western blot is shown. Densitometric values of acetylated and phosphorylated Histone H3 levels were normalized to those of total Histone H3 and the relative intensity ratios were expressed in-fold increase over time zero. Data are means ± S.E.M of four independent experiments. *P<0.05 vs control time zero. Statistical analyses performed using one-way ANOVA followed by Dunnet's ad hoc test.