Figure 5.

An IMP exists in CSF extracts. (A and B) Time course showing the ability of different APC immunoprecipitates to ubiquitinate ¹²⁵I-labeled-cyclin B 13–110 (Cyc B) in a reconstituted system containing purified E1, UBC4, and UBCx. APC was isolated from an interphase extract (APCⁱ), from a CSF extract (APC^CSF), and from a mitotic Δ90 extract (APC^m). Samples were analyzed by SDS-PAGE and phosphorimaging (B), and the ubiquitination activities were expressed as percentage of cyclin B converted into conjugates (A). (C and D) The stability of ¹²⁵I-labeled cyclin B 13–110 (Cyc B) was analyzed in APC-depleted Δ90 and CSF extracts supplemented with a mitotic APC fraction. Samples were analyzed as above (D), and cyclin B levels were quantitated (C). (E) Cdc27 immunoblot of the extracts used in C and D to control for the immunodepletion of APC. Lanes 1 and 2, Δ90 extract before and after depletion with Cdc27 antibodies, respectively; lanes 3 and 4, CSF extract before and after Cdc27 immunodepletion, respectively. A protein band from a different portion of the same blot, which nonspecifically cross-reacts with Cdc27 antisera, is shown as a loading control.