Cdc25A S82 and S88 are required for full degradation of the protein following DNA damage. HT1080 cells were transfected with FLAG-Cdc25A-wt, FLAG-Cdc25A-S82A, FLAG-Cdc25A-S88A, FLAG-Cdc25A-S82A/S88A or FLAG-Cdc25A-S88F. A) When cells were treated with 40 μM etoposide for 12 hours, FLAG-Cdc25A-wt was efficiently degraded while mutant Cdc25A S82A and mutant S88A were partially degraded. The double mutant S82A/S88A was completely resistant to degradation following DNA damage. B) Similar to FLAG-Cdc25A-S88A, FLAG-Cdc25A-S88F was partially degraded following treatment with etoposide (40 μM) for 12 hours. C) Exponentially growing cells transfected with either Flag-Cdc25A or Flag-Cdc25A-S88F were treated with cycloheximide (50 μg/mL) alone to block the protein synthesis. At the indicated time intervals cell samples were collected and Cdc25A expression was detected by western blot using Flag-M2 antibody. α-tubulin levels are shown as loading controls. Both Flag-M2 and α-tubulin monoclinal antibodies were purchased from Sigma Aldrich.