Figure 2.
In vitro activation of p34cdc2/cyclin B in wild-type BHK21 cells. Cultures of wild-type BHK21 cells synchronized in S phase with HU were incubated in the presence (CHX-treated, columns 1 and 2) or absence (non-treated, columns 3–6) of cycloheximide (10 μg/ml) for 1 h and then lysed to prepare the cytosolic extracts, as described in Materials and Methods. The cytosolic extracts were incubated at 30°C in the absence (columns 1 to 4) or presence (columns 5 and 6) of cycloheximide (10 μg/ml). Before incubation (columns 1, 3, and 5) or after incubation for 1 h (columns 2, 4, and 6), 8 μl of the extracts (protein concentration: 3 mg/ml) were mixed with the reaction-stopping buffer. p34cdc2/ cyclin B complexes were immunoprecipitated using the anti- cyclin B antibodies and assayed for histone H1 kinase activities.