Figure 3. EL and LMW-E interact with and phosphorylate Cdc25C.

A, GST pull-down assay showing the interaction of ³⁵S-labeled EL (left panel) and LMW-E (right panel) with GST-tagged fusion proteins. Ponceau-stained gel is shown below. B, 293T cells were cotransfected with Flag-tagged EL or LMW-E and c-myc-Cdc25C for 24 h. Left panel, total lysates were analyzed by western blot analysis to show expression of transfected proteins. Vinculin used as loading control. Right panel, IP was performed with monoclonal anti-Flag or a polyclonal anti-c-Myc followed by western blotting with the indicated antibodies. C, pTRE-LMW-E MCF-7 cells were synchronized with aphidicolin for 24 h, induced for 24 h, released for 4 h, and analyzed by western blotting for cyclin E, Cdc25C, and CDK1-pY15 (phospho-CDK1). Densitometric values of the Cdc25C slower migrating band is depicted in bar graph, bottom panel. D, Left panel, western blot analysis showing cyclin E expression in HeLa cells infected with control (LacZ), EL- or LMW-E–expressing adenovirus for 24 h. Middle panels, HeLa cells overexpressing cyclin E were IPed with a cyclin E antibody and kinase activity was measured using full-length GST-Cdc25A or GST-Cdc25C as substrates. The CDK2 inhibitor Roscovitine (80 μM) was added to the kinase reaction as indicated. Right panel, HeLa cells were synchronized with aphidicolin for 24 h and infected with EL or LMW-E adenovirus. Cell lysates were prepared from cells released for 6 h and subjected to a kinase assay using GST-Cdc25C 200-256 S214A or S216 A as a substrate (right panel).