Nuclear shrinking depends on conventional PKC activity. The nuclear shrinking assay was performed as in Fig. 1. Nuclear size was normalized against the HI-LEE control in A–E. (A) Kinase inhibitors were added as follows: CDK-olomoucine (2 mM), PKA-KT 5720 (56 nM), Aurora kinase–ZM 447439 (20 µM), and PKC-chelerythrine (390 µM). n = 6 different extracts. (B) Pan-PKC or rabbit IgG control antibodies were added to LEE at 2.5 µg/ml. n = 3 different extracts. (C) Calcium depletion was accomplished by supplementing LEE with 1 mM EGTA. A 45-min EGTA treatment was rescued by the addition of 4 nM CaCl2. DAG depletion was accomplished with 60 µM propranolol. n = 4 different extracts. (D) Inhibitory human PKC βI or rabbit IgG control antibodies were added to LEE at 2.5 µg/ml. n = 2 different extracts. (E) Where indicated, LEE was treated with chelerythrine (390 µM) for 45 min, then recombinant constitutively active PKC-β-ΔNPS (1 nM) was added followed by an additional 45-min incubation. n = 2 different extracts. (F) Nuclei were assembled de novo in X. laevis egg extract for 45 min. Chelerythrine (390 µM), constitutively active PKC-β-ΔNPS (1 nM), or PMA (1.6 µM) were then added, and nuclear expansion was allowed to progress for an additional 45 min. Nuclear size was normalized to the 45 min time point. n = 5 different extracts. ***, P < 0.005; **, P < 0.01; *, P < 0.05. Error bars represent SD.
