Fig. 5. CDK2 phosphorylation and cytoplasmic distribution is required for methotrexate- and docetaxel-induced cell death. (A) 293T cells were left untreated, treated with methotrexate alone (upper panel), docetaxel alone (lower panel) or given a 15 minute pretreatment with wortmannin. CDK2 was immunoprecipitated with anti-CDK2 antibody, and the phosphorylation of CDK2 was detected by immunoblotting with a phospho-Akt substrate-specific antibody. Total CDK2 was detected by a CDK2-specific antibody. (B) 293T cells were left untreated or treated with methotrexate or docetaxel, and the localization of CDK2 was detected by confocal microscopy after immunostaining with anti-CDK2 antibody followed by a Cy3-conjugated secondary antibody. (C) The cells were left untreated, treated with methotrexate alone or docetaxel alone, or the drugs were added 15 minutes after pretreatment with roscovatine. Twenty-four hours later, the percentage of cell death was assayed by flow cytometry (Nicoletti method). (D) Immortalized CDK2 wild-type (CDK2 WT), CDK2-deficient fibroblasts (CDK2^–/–), CDK2 wild-type reconstituted CDK2^–/– fibroblasts and CDK2 T39A mutant reconstituted CDK2^–/– cells were treated with either methotrexate or docetaxel, and 24 hours later the percentage of cell death was assayed by flow cytometry (propidium iodide staining).