Fig. 6. Disruption of AKAP-RII anchoring induces reversible premature chromatin decondensation. (A) Reh-RII chromatin condensed in mitotic Reh-RII extract was recovered and incubated in fresh mitotic Reh-RII extract containing 500 nM Ht31, 500 nM control Ht31-P peptide or no peptide. Percentage of PCD was monitored over 3 hours after DNA staining. (B) Chromatin (P) and reaction supernatants (S) before (Input) and after a 3-hour incubation in extract containing Ht31 or Ht31-P as in (A) were analyzed by immunoblotting using anti-AKAP95 and anti-RII mAbs. (C) Prematurely decondensed Reh-RII chromatin was recovered from Ht31-containing extract and resuspended in fresh mitotic Reh extract containing no peptide or increasing concentrations of recombinant wild-type RII or indicated mutants. Proportions of PCD were monitored after 1.5 hours by DNA staining. (D) Chromatin masses obtained at the end of incubation with wild-type RII or RII mutants were isolated and immunoblotted using anti-AKAP95 and anti-RII antibodies. (E) Reh nuclei were condensed for 2 hours in mitotic Reh extract. Condensed chromatin was recovered and incubated (T=0 hours) in mitotic extract of Reh, Reh-RII or Reh-RII(T54E) cells. The percentage of PCD was evaluated over 2 hours after DNA staining. (F) Reh nuclei were condensed for 2 hours in mitotic Reh extract as in (E). Condensed chromatin was recovered and incubated (T=0 hours) in Reh-RII mitotic extract immunodepleted for RII, or in Reh extract containing 11 nM of either wild-type RII or RII(T54E). The percentage of PCD was evaluated over 2 hours after DNA staining.