Fig. 7. Nuclear reassembly in vitro is accompanied by dissociation of RII from chromatin-bound AKAP95 and threonine dephosphorylation of RII. Mitotic Reh-RII cells were lysed by Dounce homogenization and nuclei were allowed to reform by incubating the lysate at 30°C. (A) At the indicated time points, solubilization of RII and reassembly of nuclear membranes were monitored by immunofluorescence using anti-RII mAbs (green) and affinity-purified antibodies against LBR, an integral protein of the inner nuclear membrane (red); merged images are shown (upper panels). DNA was labeled with Hoechst 33342 (lower panels). Bar, 10 µm. (B) Input (0 minutes) and decondensed chromatin fractions (75 minutes) were sedimented and pellets (P) and supernatants (S) immunoblotted using anti-AKAP95 and anti-RII mAbs. (C) Entire nuclear reassembly reactions were homogenized at the indicated time points, RII was immunoprecipitated and precipitates were immunoblotted using anti-pT antibodies. RII was also immunoprecipitated after a 75-minute control incubation of chromatin in mitotic lysate without an ATP-generating system (M 75 min).