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Figure 5 | Genome Biology

Figure 5

From: Identification of CDK2 substrates in human cell lysates

Figure 5

Phosphorylation of RL12 in vitro and in vivo. (a) HA-tagged RL12 or vector control ('vec') were transiently transfected into U2OS cells and immunoprecipitated using 12CA5 antibody. Cyclin A-CDK2 complexes were also transiently expressed separately in U2OS cells and immunoprecipitated using an antibody against cyclin A. Kinase assays were carried out using the RL12 (or control) immunoprecipitate with or without the cyclin A-CDK2 immunoprecipitate in the presence of γ-32P-ATP. (b) Similar assays were conducted using cyclin A-CDK2 and RL12 immunoprecipitates containing wild-type (wt) and the indicated RL12 phosphosite mutants. The asterisk denotes the light chain of the antibody. (c) Phosphopeptide mapping and phosphoamino acid analysis of the radiolabeled wild-type (left panel) and S38T mutant (right panel) of RL12. (d) Wild-type RL12, RL12-S38A, or vector control was transiently co-transfected with cyclin E-CDK2, catalytically inactive (dn) cyclin E-CDK2, or p21. All cells were subjected to 32P orthophosphate labeling. RL12 was immunoprecipitated from cell lysates and visualized by SDS PAGE followed by autoradiography. (e) Phosphopeptide mapping analysis was also carried out on the radiolabeled RL12 shown in (d). The bottom arrow shows a second and minor phosphorylation site detected in vivo.

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