Fig. 7

HP1β does not associate predominantly with chromatin in pluripotent cells. a No colocalization of HP1β with pericentromeric H3K9me3 foci in pluripotent cells. MEFs (left) and R1 ESCs (middle) were co-immunostained for DAPI (top), H3K9me3 (middle) and HP1β (bottom). Right panel: an enlargement of the ESC colony shown in the box. Asterisks mark examples of feeder layer MEFs used for the culture of pluripotent cells. Scale bars = 25 μm. b Chromatin immunoprecipitation (ChIP)-quantitative PCR for HP1β on major satellite repeats in MEFs and ESCs. HP1β is not enriched on major satellite repeats in pluripotent cells. The SSC144 region was used as control. Error bars represent standard error of the mean. c HP1β is predominantly nucleoplasmic/chromatin-unbound in pluripotent cells. Western blots for α-tubulin, HP1β and histone H3 in MEFs (left) and R1 ESCs (right), fractionated to the cytoplasmic fraction (S1), nucleoplasmic (nuclear chromatin-unbound) fraction (S3), and chromatin-bound fraction (P3). PonceauS protein staining in the histone range of the blot was used as a loading control (bottom). d HP1β levels in each fraction were quantified from three independent experiments. Error bars represent standard error of the mean; a.u. arbitrary units. The ratio of the nucleoplasmic fraction to the chromatin-bound fraction is indicated below for MEFs and ESCs. e Western blots for HP1γ in MEFs and R1 ESCs, fractionated to the cytoplasmic fraction (S1), nucleoplasmic (nuclear chromatin-unbound) fraction (S3), and chromatin-bound fraction (P3). Protein staining with PonceauS in the histone range of the blot was used as a loading control. The ratio of the nucleoplasmic fraction to the chromatin-bound fraction is indicated below for both MEFs and ESCs