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Figure 2


Fig. 2. Akt-mediated CDK2 phosphorylation regulates its cellular localization but is dispensable for basal CDK2 kinase activity. (A) 293T cells were transiently transfected with HA-tagged CDK2, co-transfected with Akt-CA or dominant-negative Akt (Akt-DN), or with vectors coding for CDK2-mutants. CDK2 was immunoprecipitated using anti-HA antibody and the kinase activity was assessed using histone H1 as substrate. The phosphorylated histone H1 was detected by immunoblotting with phospho-H1 antibody. The expression of wild-type CDK2 and mutant proteins was detected by anti-HA antibody. (B) The wild-type CDK2 or CDK2-T39A coding vectors were transiently transfected with either vector alone, constitutively-active Akt (Akt-CA) or Akt-DN, and CDK2 was immunoprecipitated using anti-HA antibody. The presence of cyclin A and cyclin E co-immunoprecipitated with CDK2 was detected by immunoblotting with their respective antibodies. (C) HA-tagged wild-type CDK2, CDK2 T39E or CDK2 T39A mutants were trasfected into 293T cells. The association of cyclin A and cyclin E with CDK2 was detected by western blotting after immunoprecipitating CDK2 with anti-HA antibodies. (D) Wild-type CDK2, CDK2-T39A or CDK2-T39E and Akt-CA or Akt-DN vectors were co-transfected into 293T cells. After 24 hours, the localization of CDK2 was detected by confocal microscopy after immunostaining with anti-CDK2 antibodies followed by Cy3-conjugated secondary antibody. DAPI was used to counterstain nuclei. Corresponding micrographs with nuclear and CDK2 staining were overlaid to visualize changes in CDK2 cellular localization. (E) HA-tagged wild-type CDK2 or T39 mutants were co-transfected into 293T cells with either Akt-CA or Akt-DN vectors and the association of 14-3-3 with CDK2 was determined by immunoblotting with 14-3-3 antibodies after immunoprecipitating with either HA antibody or IgG control antibody. FOXO3A interaction with 14-3-3 was used as a positive control. The input controls for CDK2, 14-3-3 and FOXO3A were shown by immunoblotting the total cell lysates with the respective antibodies.





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