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Figure 5


Fig. 5. Genetically replaced Cdk2 retains its normal subcellular localization. (A,B) Immunofluorescence staining of Cdk2 and HA-Cdk2 in serum-starved and serum-stimulated Cdk2+/+ Cdk1+/Cdk2KI MEFs at different time points. Cdk2 and HA-Cdk2 localization was detected by rabbit anti-Cdk2 and rabbit anti-HA followed by Alexa Fluor 568-conjugated goat anti-rabbit antibodies (red; Ae-h, Be-h), with the nuclei counterstained with DAPI (blue; Aa-d, Ba-d). The images were captured with a laser confocal microscope (63x). These representative pictures are from one of the three independent stainings. Scale bar: 10 µm. (C) Line graph displaying the proliferation rate of Cdk2+/+, Cdk2-/-, Cdk2+/+ Cdk1+/Cdk2KI and Cdk2-/- Cdk1+/Cdk2KI MEFs. (D) Western blots showing the expression of Cdk2, Cdk4, Cdk1, cyclin A2, cyclin B1, cyclin D1, cyclin E1, HA-Cdk2, p27 (panels 1-9 from top, respectively), HA-Cdk2/cyclin E1 and HA-Cdk1/cyclin A2 co-immunoprecipitations (panels 11 and 12 from top, respectively), as well as the histone H1 kinase activity of Cdk2 and HA-Cdk2 (Cdk2KI) (bottom two panels) in Cdk2+/+ (lane 1) Cdk2-/- (lane 2), Cdk2+/+ Cdk1+/Cdk2KI (lane 3) and Cdk2-/- Cdk1+/Cdk2KI (lane 4) MEFs. Actin (panel 10) served as a loading control.





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