Figure 3.

p47 forms a complex with p97 in mitotic cytosol. (A) Phosphorylated p47 binds to p97. His-tagged p97 (60 μg/ml), no tag p47 (30 μg/ml), or their combination was incubated with [γ-³²P]ATP, and Cdc2 (80 mU/ml) at 30°C. After 1 h incubation, His-p97 and its binding proteins were immunoprecipitated with anti-His antibodies. Phosphorylated p47 bound to His-p97 was detected by autoradiography. (B) Mitotically phosphorylated p97 binds to p47. p47 and its binding proteins were isolated from in vivo ³²P-labeled mitotic cytosol by native immunoprecipitation using anti-p47 antibodies and protein A beads. p47-binding proteins were eluted from the beads using 1 M KCl. p97 in the elution was isolated by denatured immunoprecipitation using polyclonal anti-p97 antibodies, and phosphorylated p97 was detected by autoradiography. (C) A p97/p47 complex is kept in mitotic cell extract. Synchronized and unsynchronized HeLa cells were completely homogenized until nuclear envelopes were broken and supercentrifuged to get mitotic and interphase cell extracts, respectively. Extracts were size fractionated by gel filtration, and the fractions were assayed by Western blotting with polyclonal antibodies to p97 and p47.