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. 1998 Dec 15;12(24):3882–3888. doi: 10.1101/gad.12.24.3882

Figure 8.

Figure 8

Phosphorylated p21 is a potent Cdk inhibitor. Phosphorylated p21 was obtained by incubating the inhibitor with an excess of activated cyclin A/Cdk2 and 1 mm ATP. Phosphorylated (p21-P) and mock-incubated, nonphosphorylated p21 (p21) were purified and immobilized on protein A–Sepharose beads as described in Fig. 2. These beads and anti-cyclin A antibodies bound to control beads were incubated with 250 ng of active cyclin A/Cdk2 complex and washed and equal aliquots were analyzed for associated kinase activities (histone H1 kinase) and the amount of precipitated proteins. Precipitated proteins were detected after SDS-PAGE and Western blotting and analyzed for precipitated kinase subunit Cdk2 (Cdk2, α-PSTAIRE) and inhibitor p21 (p21, α-p21). The phosphorylated forms of p21 show a lower electrophoretic mobility compared to nonmodified p21. Precipitated kinase activities associated with these samples were determined in assays using [γ-32P]ATP and histone H1 as substrate and detected after SDS-PAGE by autoradiography of the dried gel (histone H1 kinase).

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