Figure 5

Cdc25A S88F mutant retains its phosphatase activity both in vitro and in vivo. A) GST-Cdc25A-wt, GST-Cdc25A-S88A and GST-Cdc25A-S88F were assayed by hydrolysis of pNPP in vitro. No major effect on Cdc25A mutants was observed compared with wildtype. B) HT1080 cells were transfected with GFP-Cdc25A-WT, GFP-Cdc25A-S88F or GFP-H2B control; 12 hours post transfection, lysates from the transfected cells were assayed for Cdc2-Y15 phosphorylation. Cells equally transfected with either wildtype or mutant Cdc25A show similar levels of Y15 phosphorylation 12 hours post transfection. Anti-GFP antibody (a mixture of two monoclonal antibodies) was purchased from Roche Applied Sciences. Cdc2 and Cdc2-Y15 antibodies were purchased from Santa Cruz Biotech. (Santa Cruz, CA).