FIG. 4.
Phosphatase treatment of AHisp27 converts it to an inhibitory form. (A) Ap27 and G0p27 were treated with PAP (lanes 2 and 4) or mock treated (lanes 1 and 3) and then repurified. Immunoblot analysis was performed with p27 antibodies (W). (B) Recombinant cyclin D-cdk4 was incubated with Ap27, PAP Ap27, G0p27, or PAP G0p27 (lanes 1 to 4) and immunoprecipitated (Ip) with p27 antibodies to detect cdk4 association by immunoblot analysis. Normal rabbit serum (NRS) served as a control. (C) Cyclin D-cdk4 (DK4) was mock incubated (lane 1) or incubated with Ap27, G0p27, PAP Ap27, and PAP G0p27 (lanes 2 to 5 and 7 to 10). Recombinant cyclin H-cdk7 (cdk7) in the presence of [γ-32P]ATP was added (+) or not added (−). Samples were immunoprecipitated with cdk4 antibodies to detect radiolabeled cdk4 (cdk4*). (D) Recombinant cyclin D-cdk4 was incubated with [γ-32P]ATP and cyclin H-cdk7 (lanes 2 and 3). PAP Ap27 was added to half of this phosphorylated cyclin D-cdk4, followed by immunoprecipitation (Ip) with cdk4 antibodies to detect cdk4 phosphorylation (cdk4*) (lanes 2 and 3).