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Fig. 1. MCM5 specifically interacts with the wild-type CLS of cyclin E. (A) Endogenous cyclin E co-immunoprecipitates MCM5. Lysates from asynchronous HeLa S3 cells were subjected to immunoprecipitation, as described in the Materials and Methods, with anti-cyclin E antibody or control IgG, as indicated, and immunoprecipitates were western blotted with anti-MCM5 and -cyclin E antibodies. (B) Cyclin E directly interacts with full-length MCM5. MCM5 was radiolabeled with [³⁵S]methionine in an in vitro transcription-translation system as described in the Materials and Methods. The radiolabeled protein was incubated with glutathione-agarose beads bound to GST or GST–cyclin E. Beads were washed, eluted with SDS-PAGE sample buffer and electrophoresed on 10% gels. Left: Coomassie-stained gel of 25% of GST and GST–cyclin E. Right: an autoradiograph of MCM5 is shown. (C) Mutation of the cyclin E CLS disrupts MCM5–cyclin E interaction. Flp-In T-Rex CHO cells were induced to express the indicated Myc-tagged cyclin E constructs as described in the Materials and Methods. Lysates were prepared and anti-Myc immunoprecipitates were electrophoresed on 10% SDS gels, followed by western blotting for MCM5 (top panel), Myc (middle panel) and Cdk2 (lower panel).

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