FIG. 1.

PAP binds cyclin B₁ in vivo and in vitro. (A) PAP associates with cyclin B₁ in vivo. Coimmunoprecipitation and Western blot analysis of Sf9 insect cell extracts made from cells infected with recombinant baculoviruses expressing either PAP plus cyclin B₁ or cyclin B₁ alone. Lysates were immunoprecipitated with a polyclonal antibody raised against PAP. The immunoprecipitates (lanes 1 and 2) and 10% of the cell extracts used (lanes 3 and 4) were subjected to SDS-PAGE and subsequent Western blot analysis using a monoclonal antibody against human cyclin B₁. The position of cyclin B₁ is indicated. (B) PAP binds cyclin B₁-cdc2 directly. Purified PAP II (1 μg; strips 1 and 2), purified p53 (1 μg; strip 3), and bacterial whole-cell extract expressing human cyclin B₁ (strip 4) were first immobilized on nitrocellulose. Following renaturation by serial dilution with guanidine-HCl, strips 1, 3, and 4 were incubated with purified cyclin B₁-cdc2 (100 ng). After washing, cyclin B₁ was detected by immunoreactivity to the anti-cyclin B₁ antibody. An arrow on the left indicates the position of PAP II. An arrow on the right indicates the position of cyclin B₁.