Mammalian IMPACT forms a complex with Cdc28 and CDK1.

<p><b>(A)</b> Mammalian IMPACT expressed in yeast precipitates Cdc28. Two different transformants of <i>yih1Δ</i> strain (BY4741) expressing either GST-IMPACT or GST alone from a galactose inducible promoter were grown to log phase in SGal. WCEs were prepared and equivalent amounts of protein (1 mg) were subjected to GST-pull-down assays. The precipitated complexes were analyzed by immunoblot for the indicated proteins. The input lanes (left-panel) contained 4% of the WCEs used in the assay. <b>(B)</b> CDK1 co-precipitates with Flag-tagged IMPACT in N2a cells. Undifferentiated mouse N2a cells were transfected with a plasmid expressing IMPACT fused to Flag or with the vector alone (pFLAG). Cell lysates were cleared with protein-A agarose and subjected to immunoprecipitation with anti-Flag antibodies (M2-Flag-Resin). All the precipitated material and 1% of the input material were subjected to immunoblot to detect Flag-IMPACT, CDK1, and GAPDH as negative control.