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Figure 1 | Genome Biology

Figure 1

From: Identification of CDK2 substrates in human cell lysates

Figure 1

Characterization of the engineered CDKs. (a) Cyclin E-CDK2/3 complexes, or their F80A engineered counterparts (indicated by asterisks), were immunoprecipitated from transfected U2OS cell lysates via the HA-tag on the CDK subunit and subjected to in vitro kinases assays with 10 μM of either normal ATP or PE-ATP analogue. Phosphorylation of GST-Rb was monitored by immunoblotting with a phosphospecific anti-pS780-Rb antibody (New England Biolabs). The wild-type kinases cannot use PE-ATP. (b) Thin-layer chromatography - analysis reveals hydrolysis of ATP in cell lysate and transfer to acceptor nucleotides (left). The positions of the free phosphate and ATP are indicated. In contrast, ATP-γ-S is not hydrolyzed in cell lysates (right). (c) Kinase assays were performed using wild-type and cyclin A-CDK2 (F80A) complexes purified from E. coli and GST-Rb in the presence of 200 μM of ATP, ATP-γ-S and PE-ATP-γ-S at room temperature for 2 h. Kinase reactions were analyzed by SDS gel electrophoresis and visualized by Coomassie staining. Extent of the GST-Rb phosphorylation was monitored by the electromobility shift of GST-Rb.

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