Figure 2.
Biochemical characterization of the response of somatic cell extracts to His6-cyclin B
(A) Autoradiogram of a dried gel containing radioactively phosphorylated histone H1 (top). His6-cyclin B was added to somatic cell extracts at the indicated concentrations and incubated for 20 min at room temperature. Kinase assays were performed to determine the amount of P32 incorporated into the CDK1 substrate histone H1. Quantification (n=2–9) is shown (bottom) with error bars representing ± one standard deviation. The coefficient of variation was constant at 30%.
(B) The effect of cyclin B on cellular proteins was analyzed by immunoblotting (phospho-CDK1, CDC25 and actin as a loading control) or by adding radiolabeled in vitro translated proteins to the extract (WEE1 and securin).
(C) Gel filtration of extracts following His6-cyclin B addition. Following a 20 min incubation at room temperature with the indicated cyclin B concentrations, extracts were loaded onto a Superdex 200 column, and fractions were analyzed by immunoblotting for CDK1, cyclin B, and WEE1. Shown are apparent molecular weight positions.
(D) Cyclin B-CDK1 binding to mitotic regulators. His6-cyclin B bound to Ni-NTA agarose was added to the extract at a concentration below (100 nM) or above (2000 nM) the level for robust CDK1 activity. Following a 30 min incubation, the extract was centrifuged and the Ni-NTA beads were collected. The beads were washed three times with extract dialysis buffer and the bound proteins were analyzed by SDS-PAGE and immunoblotting.