Fig. 5. Effect of exogenous PTEN expression on GST-Grp1-PH labeling density after PDGF stimulation in U87MG cells. Ultrathin cryosections of both untransfected and GFP-PTEN-transfected U87MG cells, stimulated with 50 ng/ml PDGF for 10 minutes, were labeled with GST-Grp1-PH. (A) The labeling density over the plasma membrane and endoplasmic recticulum (ER) compartments in cells expressing PTEN [WT] is shown (densities obtained as described in Fig. 2 legend). (B) Nuclear GST-Grp1-PH labeling of cells transfected with PTEN [WT] is expressed as the number of gold particles related to area. Results are the means from a single experiment in which 616 intersections (PM and ER) or 1614 gold particles (nucleus) were counted. Error bars in A and B represent s.e.m. (C) The GST-Grp1-PH labeling for plasma membrane, ER and nuclear compartments in cells transfected with various PTEN constructs (WT; C124S phosphatase dead; or PTEN nls, containing an engineered nuclear localization signal). Data are normalized labeling densities from a representative data set of two similar experiments. (D) The subcellular localization of GFP-PTEN and GFP-NLS-PTEN in U87MG cells. Whereas GFP-PTEN is observed throughout the cytoplasm and nucleus, GFP-NLS-PTEN appears to be exclusively localized to the nucleus. (E) The effects of increasing expression of GFP-PTEN and GFP-NLS-PTEN on pS473-PKB phosphorylation in U87MG cells. The expression of each construct was titrated by adding increasing volumes of baculovirus supernatant to cells (0-1.0 ml in 10 ml of medium). After 24 hours, cells were lysed and the PTEN expression and pS473-PKB phosphorylation analysed by western blotting. The blots shown are representative of three independent experiments.