Figure 7.

Inhibition of the MAPK pathway maintains radiation-induced Cdc2 tyrosine 15 phosphorylation 24 h after irradiation. A431-TR25-EGFR-AS cells were pretreated for 30 min before irradiation with a MEK1/2 inhibitor (PD98059, 10 μM) or DMSO control. Cells were irradiated (2 Gy), and the phosphorylation and activity of Cdc2, and the total expression of Cdc2 and Cdc25C, were determined at the indicated times after irradiation. For kinase activity, cells were lysed, and portions (∼100 μg) from each plate were subjected to immunoprecipitation, followed by immune complex kinase assays with the use of histone H1 as substrate. Results from a representative experiment (±SEM) are shown (n = 3); *p < 0.01 less than control value. (Inset) The phosphorylation of Cdc2 tyrosine 15 was determined after Cdc2 immunoprecipitation 6 and 24 h after irradiation. Cells were lysed, and portions (∼100 μg) from each plate were subjected to immunoprecipitation for Cdc2. Immunoprecipitates were resolved via SDS-PAGE, followed by immunoblotting to determine Cdc2 tyrosine 15 phosphorylation levels. Exposure time was 5 min. The total protein levels of Cdc2 and Cdc25C were also determined by immunoblotting 24 h after radiation exposure. Cells were lysed, and equal portions (∼100 μg) from each plate were subjected to SDS-PAGE, followed by immunoblotting to determine Cdc2 and Cdc25C protein levels. Exposure time was 4 min. Results of a representative experiment for each condition are shown (n = 3).