Figure 2.

Set7/9 controls Cdk2 activity by affecting the expression of cyclin E. (a) U2-OS parental control cells and cells with knockdown expression of Set7/9 and p53 were treated with doxorubicin for the indicated times and subject to immunoprecipitation with anti-cdk2 or anti-cdk4 antibodies. The resulting immunopurified Cdk2/cyclin E and cdk4/cyclin D complexes were analysed for their ability to phosphorylate H1. (b) Western blot analysis of the cell lines indicated above for the levels of expression of Set7/9, cdk2, cyclin E, E2F1, and p53. Actin signal was used as a loading control. (c) Western blot analysis of U2-OS and U2-OS Set7/9⁻ cells (Set7/9KD) treated with doxorubicin for the indicated times and subject for immunoblotting with cdk4, cyclin D1, and cyclin E antibodies. Ku70 was used as a normalisation control. (d) Tet-on U2-OS cells with inducible expression of control shRNA or shRNA against Set7/9 were induced with 2 μg/ml doxycycline or tetracyclin (Tet) for 3 days before assessing the efficiency of knockdown of Set7/9 by western blotting. Actin signal was used as a loading control. (e) The abovementioned cells were treated with doxorubicin and analysed for the levels of cyclin E and E2F1 by western blotting