Figure 4

Protein phosphatase activity of PTEN (A) In order to determine the specificity of the PTEN phosphatase assay, PTEN-negative U87 glioblastoma cells were transiently transfected with pCMV-PTEN expression vector (PTEN) or with pCMV-blue vector without PTEN cDNA insert (Vector). As a further control, non-transfected U87 cells were used in parallel (U87). 24 hours after transfection, cellular lysates were harvested and subjected to immunoprecipitation with PTEN specific monoclonal antibodies. The collected antigen was assayed for tyrosine phosphatase activity as described in Materials and Methods. Shown is the amount of radiolabeled phosphate released from the substrate (mean of three experiments the quantity of phosphate released in the absence of added antigen. (B) In order to determine PTEN phosphatase activity in suspension cells, MDAH cells were transferred to suspension culture conditions for the times indicated. Total cellular lysates were prepared and subjected to immunoprecipitation with PTEN specific monoclonal antibodies. The collected antigen was assayed for tyrosine phosphatase activity as described in Materials and Methods. Shown is the amount of radiolabeled phosphate released from the substrate (mean of three experiments ± sd). released in the absence of added antigen.