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Fig. 3. S10 and S28 phosphorylation are targeted to distinct pools of histone H3. (A) Crosslinked chromatin was prepared from quiescent C3H 10T
fibroblasts which were stimulated with 25 ng/ml anisomycin for 30 minutes (even-numbered lanes) or left untreated (odd-numbered lanes). Chromatin was immunodepleted with anti-phosphoS10-H3occ (lanes 5, 6), anti-phosphoacetyl-H3 (lanes 7, 8) or anti-phosphoS28-H3 (lanes 11, 12), or was mock immunoprecipitated in the absence of antibody (lanes 3, 4). Immunodepleted material and corresponding undepleted (input) samples (lanes 1, 2 and 9, 10) were analysed on 15% SDS-PAGE gels and western blotted with anti-phosphoS10-H3occ (panel i), anti-phosphoacetyl-H3 (panel ii) or anti-phosphoS28-H3 (panel iii). Before blotting, membranes were stained with Ponceau-S to show that no overall loss of bulk histones occurred by immunoprecipitation (data not shown), consistent with phosphorylation being targeted to a small fraction of total H3. (B) Quiescent C3H 10T fibroblasts were stimulated with 25 ng/ml anisomycin for 45 minutes (panels iv-ix) or left untreated (panels i-iii). Cells were fixed and histone H3 phospho-epitopes were detected by immunofluorescence as described in Materials and Methods. Primary antibodies used were: anti-phosphoS10-H3 (panels i, iv, vii) and anti-phosphoS28-H3 (panels ii, v, viii). Anti-rabbit-IgG-Alexa-488 and anti-rat-IgG-Cy3 were used as secondary antibodies. Images were collected with a confocal laser scanning microscope. Alexa-488-(green) and Cy3-(red) fluorescence images are shown and merge images are presented (panels iii, vi, ix). Images from control and stimulated cells were acquired with identical image collection protocols. Enlarged regions from stimulated nuclei are presented (inset, panels iv-ix). Scale bar, 4 µm. (C) sAn-stimulated nuclei were prepared for immunofluorescence and labelled with anti-phosphoS10-H3 and anti-phosphoS28-H3 antibodies as in (B). Lines were drawn at random through the nucleoplasm profiles of intensity for both the green (anti-phosphoS10-H3; green line) and red (anti-phosphoS28-H3; red line); channels were plotted (y-axis; arbitrary units) against the position along the line (x-axis; voxels). (D) CCF analysis was performed for anti-phosphoS10-H3 and anti-phosphoS28-H3 co-stained cells (solid line), and for cells labelled with anti-phosphoS10-H3 (non-occ.) detected by red- and green-fluorescent antibodies (broken line), as described in Materials and Methods. Two nuclei were chosen from independent experiments and for each nucleus, three independent line-scans were taken as in (C) for CCF analysis (six line-scans in total). CCF analysis produces a graph showing the dependency of the correlation between the fluorescent profiles of the red and green channel (calculated as Pearson's coefficient, RP, y-axis) on the shifting of the intensity profiles with respect to one another (
X, x-axis). Each CCF represents the mean of the six line-scans processed.
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