title: Methods to Study PTEN in Mitochondria and Endoplasmic Reticulum.
abstract: Although PTEN has been widely described as a nuclear and cytosolic protein, in the last 2 years, alternative organelles, such as the endoplasmic reticulum (ER), pure mitochondria, and mitochondria-associated membranes (MAMs), have been recognized as pivotal targets of PTEN activity.Here, we describe different methods that have been used to highlight PTEN subcellular localization.First, a protocol to extract nuclear and cytosolic fractions has been described to assess the "canonical" PTEN localization. Moreover, we describe a protocol for mitochondria isolation with proteinase K (PK) to further discriminate whether PTEN associates with the outer mitochondrial membrane (OMM) or resides within the mitochondria. Finally, we focus our attention on a subcellular fractionation protocol of cells that permits the isolation of MAMs containing unique regions of ER membranes attached to the outer mitochondrial membrane (OMM) and mitochondria without contamination from other organelles. In addition to biochemical fractionations, immunostaining can be used to determine the subcellular localization of proteins; thus, a detailed protocol to obtain good immunofluorescence (IF) is described. The employment of these methodological approaches could facilitate the identification of different PTEN localizations in several physiopathological contexts.
authors.affiliation: Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology and LTTA center, University of Ferrara, Via Fossato di Mortara 70 c/o CUBO, 44121, Ferrara, Italy.
authors.forename: Sonia
authors.initials: S
authors.lastname: Missiroli
authors.affiliation: Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology and LTTA center, University of Ferrara, Via Fossato di Mortara 70 c/o CUBO, 44121, Ferrara, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Morganti
authors.affiliation: Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology and LTTA center, University of Ferrara, Via Fossato di Mortara 70 c/o CUBO, 44121, Ferrara, Italy.
authors.forename: Carlotta
authors.initials: C
authors.lastname: Giorgi
authors.affiliation: Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology and LTTA center, University of Ferrara, Via Fossato di Mortara 70 c/o CUBO, 44121, Ferrara, Italy. pnp@unife.it.
authors.forename: Paolo
authors.initials: P
authors.lastname: Pinton
mesh_terms: D000818:Animals; D003584:Cytological Techniques / Q000379:methods*; D004721:Endoplasmic Reticulum / Q000737:chemistry*; D005453:Fluorescence; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D008928:Mitochondria / Q000737:chemistry*; D051059:PTEN Phosphohydrolase / Q000032:analysis*
chemical_list: D051059:PTEN Phosphohydrolase
keywords: Endoplasmic reticulum; Immunofluorescence; Mitochondria; Mitochondria-associated membranes (MAMs); Nuclear extraction; PTEN; Subcellular fractionation