Figure 1.

DNA damage induces the phosphorylation of pRB at Ser612. (A–C) Ser612 but not other Cdk phosphorylation sites is phosphorylated after DNA damage. MCF7 cells were exposed to 30 Gy of γ irradiation (IR) (A), 10 nM actinomycin D (ActD) (B), or 10 J/m² of UV radiation (UV) (C) for the indicated periods. Cell lysates were immunoprecipitated with anti-pRB antibodies and Western blotting was performed. (D) Analysis of Cdk2 and Cdk4 kinase activities after DNA damage. MCF7 cells were exposed to 10 nM ActD for the indicated periods. Cell lysates were immunoprecipitated with anti-Cdk2 or anti-Cdk4 antibody, and an in vitro kinase assay was performed by using recombinant pRB as a substrate. The reaction products were analyzed by immunoblotting using anti-phospho-Thr821 (for Cdk2 activity) or anti-phospho-Ser608 (for Cdk4 activity) antibodies.