Fig. 5. Analysis of demethylation-induced chromatin remodeling in demethylated ES cells. (A) Outline of chromatin fractionation and analysis methods. Nuclei were isolated from ES cells and chromatin was digested in situ with micrococcal nuclease. Nucleosomes were then driven from nuclei by two successive hypotonic treatments (extractions), which liberate free nucleosomes into the supernatant. Thereafter, DNA and protein moieties of isolated nucleosomes were analyzed. (B) Micrococcal nuclease-digested DNA isolated from J1, B9 and C/C cells after initial and second hypotonic extractions. (C) Protein extracted from the same chromatin fractions shown in B detected by Coomassie Blue staining. (D) Western analysis of chromatin proteins in the same preparations shown in B and C. (Loading is identical to Coomassie Blue-stained gel in C.)