FIG. 7.
Altered pericentric heterochromatin in Rb1ΔL/ΔL fibroblasts. (A) Representative fluorescent micrographs of interphase nuclei stained with DAPI and antibodies against trimethylated H4-K20 are shown in the left column. Similarly stained unfixed chromosome spreads for wild-type, Rb1ΔL/ΔL, and Rb1−/− are shown on the right. The arrow indicates joined centromeres. (B) Chromatin immunoprecipitations were performed using the antibodies indicated below the gel. Major satellite repeat sequences were PCR amplified and analyzed by ethidium bromide staining of an agarose gel (a negative image of the gel is shown). PCR amplification of these repeats generated both the 74-bp and 308-bp bands. Labels above each gel lane indicate the genotype of chromatin used for that lane. IgG, immunoglobulin G. (C) Major satellite sequences in precipitated chromatin were also analyzed by real-time PCR to quantify differences between wild-type, Rb1−/−, and Rb1ΔL/ΔL. The error bars indicate 1 standard deviation from the mean. The asterisk indicates a statistically significant difference (t test; P < 0.001).