FIG. 3.

Binding of Cks proteins to monomeric cdks. (A) Binding of p34^cdc2 and cyclin B to CksHs2-Sepharose beads. Binding of in vitro-translated, ³⁵S-labeled p34^cdc2 or luciferase (as a control) to either ovalbumin (ova)-Sepharose beads or CksHs2-Sepharose beads was assessed by SDS-PAGE and phosphorimage quantitation. Values represent the means of five experiments with the indicated standard errors. Binding of GST-cyclin B or GST to either ovalbumin-Sepharose beads or CksHs2-Sepharose beads was assessed by immunoblotting with antibody specific for GST and quantitation by phosphorimage analysis. The GST was added at 100-fold higher concentration (3.5 μM) than the GST-cyclin B. Values for GST-cyclin B binding represent the means of three experiments. The standard errors were too small to show on this graph. (B) Binding of CksHs2 to monomeric p33^cdk2. A high concentration of immunoprecipitated HA-p33^cdk2 was incubated with 11 μM partially purified CksHs2 and then pelleted and washed. Immunoprecipitates were analyzed by SDS-PAGE followed by immunoblotting with an anti-Cks antibody. A control for background precipitation of CksHs2 was performed by substituting buffer for p33^cdk2 (lane 1).