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Fig. 2. Differential stability of HP1 assemblies to detergent extraction and RNase digestion. (A) Partitioning of HP1 proteins after permeabilization with Triton X-100 and subsequent treatment with RNase A, as assessed by western blotting. Lanes: W, whole cell lysate; Pt, insoluble residue; S1, material released by Triton; S2, material released by the subsequent RNase digestion. (B) Distribution of the nucleolar protein B23 (control) after treatment with Triton alone or Triton followed by RNase. Note that the protein is not removed from the nucleoli by the detergent alone, but is fully removed after a combined Triton and RNAse treatment. Only merged images are shown (green: antibody staining; red: PI). (C-E) Distribution of HP1 proteins under conditions similar to those described in (B). A gallery of merged images in `projection' mode are shown on the left (green: antibody staining; red: PI), and selected and highly magnified sections are depicted on the right. Arrows indicate the cells from which these sections were taken and asterisks denote the position of nucleoli. (F) In situ binding of recombinant HP1 proteins (0.25 µg/ml) to Triton-ghosts and Triton/RNase A-ghosts. For each protein, antibody staining (anti-GST) is presented on the left, and the merged image after counter-staining with PI is depicted on the right. The profiles are of C127 cells. Analogous experiments with HeLa cells yield similar results. Bar, 5 µm.
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