Figure 6. SCML2 interacts with CDK2/CYCE2 complexes in G1 and regulates their activation.

(A) Western blot analysis of the SCML2B pull-down in 293 cells expressing FS-SCML2B, from asynchronously growing cells (As), and cells treated with thymidine (Thy) or nocodazole (NCZ). The pull-down was analyzed using SCML2, CDK2, CYCE2, and BMI1 antibodies. Shown on the right is 1.5% of the input. (B) Immunoprecipitation of CDK2 in cells treated with a control siRNA (C) or with siRNA specific for SCML2 (si #2) or p21 (p21), arrested in mitosis with nocodazole and released for different times, as indicated. Two percent of the input is shown (left) along with the elution (right). (C) Densitometric analysis of the amount of SCML2 pulled down by CDK2 at different time points after exit from mitosis, as in (B); the percentage of pull-down was normalized versus the input material and related to the amount of CDK2 in the pull-down. (D) Quantification of the kinase activity of CYCE2-associated complexes pulled down from control-treated U2OS cells and cells treated with siRNA #2 for SCML2, either growing asynchronously (As) or at different times after release from arrest in mitosis with nocodazole, as indicated. The reaction was performed in replicates, and the experiment was repeated twice. The levels of H1T146Ph were measured by Western blot with specific antibodies, and the levels of CYCE2 and p21 in the reaction are also shown.