Fig. 5.

Ubiquitylation of p57^Kip2 by the recombinant SCF^Skp2 complex in vitro. (A) The recombinant SCF^Skp2 complex purified from Sf21 insect cell lysates was analyzed by SDS/PAGE and staining with Coomassie brilliant blue. The positions of the various epitope-tagged recombinant proteins are indicated. (B-D) The recombinant SCF^Skp2 complex was assayed for the ability to mediate the ubiquitylation of p57^Kip2 (B), p27^Kip1 (C), or HIF-1α (D) in the presence of ATP, Uba1, UbcH5A, and GST-ubiquitin (Ub), and in the absence or presence of the recombinant cyclin E-CDK2 complex or Cks1. Reaction products were subjected to immunoblot analysis with Abs to p57^Kip2 (B), p27^Kip1 (C), or HPC4 (D).