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. 2010 Oct 8;11(11):876–882. doi: 10.1038/embor.2010.153

Figure 5.

Figure 5

Cyclin-dependent kinase 9 interacts with components of the replication stress response, localizes to chromatin and reduces chromatin-bound RPA after replication stress. (A) Cells were transfected with CDK9–HA, harvested to prepare nuclear extracts and immunoprecipitated with HA or control IgG antibodies. Bound proteins were washed, separated by SDS–PAGE and immunoblotted with antibodies against ATR, Claspin, ATRIP, HA, RPA32, and PCNA. (B–D) Endogenous CDK9, ATR, Claspin or ATRIP were immunoprecipitated from cell lysates as indicated. Immunocomplexes were washed, separated by SDS–PAGE and immunoblotted with antibodies against the indicated proteins. (E) U2OS cells were transfected with the indicated siRNAs and tested for sensitivity to HU. Mean and s.d. values from three replicate experiments is shown. (F) Soluble and chromatin fraction of U2OS cells transfected with NT or CDK9-3 siRNA for 72 h and treated with or without 3 mM HU for 20 h. Chromatin-bound RPA70/ORC2 and CDK9/ORC2 ratio of representative blot from three independent experiments is shown. APH, aphidicolin; ATR, ataxia telangiectasia and Rad3-related protein; ATRIP, ATR-interacting protein; CDK9, cyclin-dependent kinase 9; HA, haemagglutinin; HU, hydroxyurea; IB, immunoblotted; IR, ionizing radiation; ORC2, origin recognition complex 2; PCNA, proliferating cell nuclear antigen; RPA, replication protein A; SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis; siRNA, small interfering RNA.