Fig. 6. Characterization of Pds5A and Pds5B in Xenopus egg extracts. (A) Pds5A (lane 2) and Pds5B (lane 3) were immunoprecipitated from Xenopus HSS with the corresponding antibodies. Cohesin complexes were immunoprecipitated in parallel reactions with anti-Smc3 (lane 4), and non-immune rabbit IgG was used as control (lane 1). The immunoprecipitates were analyzed by immunoblotting. (B) Sperm chromatin was incubated in interphase LSS to assemble interphase nuclei and then half a volume of CSF (mitotic) LSS was added to trigger entry into mitosis. Chromatin fractions were isolated at the times indicated (lanes 3-13), and analyzed by immunoblotting. A sample from a mock assembly reaction without sperm chromatin (lane 2) and an aliquot of the interphase extract were also included (lane 1). (C) Interphase chromatin was assembled in interphase LSS that had been immunodepleted with non-immune IgG (lanes 1 and 4; mock-dep), anti-Pds5B (lanes 2 and 5; Pds5B-dep) or a mixture of anti-Smc1 and anti-Smc3 (lanes 3 and 6; cohesin-dep). Aliquots of each extract (lanes 1-3) and the corresponding chromatin fractions (lanes 4-6) were analyzed by immunoblotting.