Figure 3.

Plx1 and aurora B phosphorylate different substrates in vitro. (A) An immobilized fraction of interphase cohesin was mixed with a soluble fraction containing cdc2–cyclin B (lane 1), Plx1 (lane 2), or XAUB (lane 3) in the presence of [γ³²P]ATP. Following incubation for 1 h, the beads were washed and the bound cohesin fractions were analyzed by SDS-PAGE followed by autoradiography. A single lane of the silver-stained gel is also shown (left). (B) Chromosomes were assembled in mock-depleted (lanes 1,3,5) and aurora B-depleted (lanes 2,4,6) mitotic HSS in the presence of [γ³²P]ATP, isolated in a sucrose cushion and fractionated by SDS-PAGE. The gel was stained with silver (lanes 1,2) and incorporation of the radioactive label was detected by autoradiography (lanes 3,4). Chromosomes were also assembled in parallel reactions without [γ³²P]ATP and analyzed by immunoblotting with anti-phosphoH3 antibody (lanes 5,6).