Figure 2
From: Characterization of a novel PTEN mutation in MDA-MB-453 breast carcinoma cell line
Biochemical and biological properties of PTEN E307K mutant. (A) The phosphatase activity of the indicated PTEN isoforms was measured in the presence or absence of PIP2 using the malachite green phosphatase assay with PIP3 as the substrate. (B) Vector control (C), WT, E307K, and ΔCat expression plasmids were transfected in 293T cells. PTEN proteins were immunoprecipitated with an anti-AU5 antibody and immobilized on sepharose beads. Phosphatase activity was measured in triplicates using the malachite green assay with PIP3 as the substrate. The relative levels of PTEN isoforms in the immunoprecipitates were monitored by a polyclonal anti-PTEN antibody (insert). Similar data was reproduced in an additional experiment. (C) Vector control (C), WT, and E307K cDNAs were transduced into Pten -/- MEFS (left panel) and U87MG (right panel) cells through retroviral-mediated gene transfer. Puromycin (1 μg/ml) resistant mass cultures were analyzed for relative proliferative capacity after 6 days in cultures. The fold-increase in cell numbers was measured by cell counting. Data represents triplicated measurements from a single experiment. Bars, SD. *, p < 0.05. ***, p < 0.0005.