Measurement of PTEN activity in vivo by imaging phosphorylated Akt.
This chapter describes an indirect approach to measure PTEN's lipid phosphatase activity in vivo. PTEN counteracts phosphatidylinositol 3-kinase action in dephosphorylating 3-phosphorylated phosphoinositides. Therefore, PtdIns(3,4,5)P3-dependent activation and phosphorylation of the survival kinase Akt can be used as readout for cellular PTEN activity. Here we have outlined a detailed procedure employing a phosphoserine-specific anti-Akt antibody to examine the content of phosphorylated Akt by immunofluorescence and its dependence on PTEN activity.
Rosivatz
Erika
E
Division of Cell and Molecular Biology, Imperial College London, London, UK. e.rosivatz@imperial.ac.uk
Woscholski
Rudiger
R
D016475:3T3 Cells; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D051379:Mice; D008856:Microscopy, Fluorescence; D051059:PTEN Phosphohydrolase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000032:analysis* / Q000276:immunology / Q000378:metabolism*; D013194:Staining and Labeling
D000906:Antibodies; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D051059:PTEN Phosphohydrolase